EP1427850A2 - Human toxicologically relevant genes and arrays - Google Patents

Abstract

The invention provides for a set of toxicologically relevant human genes which may be used to predict toxicological responses on a cellular, organ, or system level. Methods of identifying, selecting, isolating, and evaluating toxicologically relevant human genes are provided. Arrays comprising human genes that are useful for toxicological screening of drugs, pharmaceutical compounds, or chemicals are also provided. The human array provides a method by which toxicological responses can analyzed in a rapid and efficient manner. Methods of identifying and isolating human genes for a human array are also provided. Further, methods of making a human gene array and using a human gene array are disclosed. These methods are also useful for isolating human genes and for discovery of novel human genes. Also provided is a database of human toxicological responses which are useful for predicting toxicological response to agents on a cellular level, organ-by-organ, and system-by-system. Methods of obtaining such databases containing human toxicological data and methods of use are also provided herein. Further, algorithms which allows for evaluation and/or 1 analysis of toxicological data are also provided herein.

Description

HUMAN TOXICOLOGICALLY RELEVANT GENES AND ARRAYS

TECHNICAL FIELD

[0001] This invention is in the field of toxicology. More specifically, the invention provides for methods to identify and isolate human genes which are indicative of toxicological responses, human genes which can be used to determine toxicological responses in vitro and in vivo to various agents, methods of making human microarrays, and methods of using human microarrays.

BACKGROUND OF THE INVENTION [0002] Every year, many new drugs and chemical compounds are discovered, produced, and introduced into the public domain. Guidelines set forth by the U.S. Food and Drug Administration (FDA) require toxicity studies to be conducted before a new drug or compound can be approved for human consumption or use. Toxicological studies are an important part of drug development but toxicity studies traditionally have required long time periods using a variety of animal models, and are quite often very expensive to conduct. Additional information on safety and toxicity is obtained during clinical trials on humans which are also time consuming and can require long time periods to conduct. A two year toxicity study in rats can cost approximately $800,000. See, for example, Casarett andDoull's Toxicology, 4th Edition, M.O. Amdur et al., eds. Pergamon Press, New York, N.Y. p. 37 (1991). Further, traditional toxicology studies are not practical for assessing the toxicity ofthe large numbers of drug candidates derived from high throughput screening of combinatorial chemical libraries. Traditional toxicological methods have offered little insight into molecular mechanisms of toxicity, which makes extrapolation of toxicity results from animal models to humans difficult and often result in numerous failures in subsequent stages of development and post-launching ofthe drug. [0003] Toxicogenomics allows for a better understanding of mechanisms of organ and system toxicity and facilitates prediction of deleterious outcomes prior to their detection by more laborious and time-consuming means. If toxicity manifested at the organism level is preceded by altered expression of related genes, then detection of altered gene expression may serve as an early warning for subsequent deleterious outcomes. Altered gene expression may precede organ or system outcomes by weeks, months or even years. To the extent that a causal or predictive relationship can be demonstrated between early alterations in gene expression and delayed manifestations of toxicity, measuring the alterations in gene expression may reduce reliance on observing delayed manifestations of toxicity. Better understanding of molecular mechanisms through toxicogenomics may also improve the predictive accuracy of animal models to humans, and in vitro systems to in vivo settings. A molecular approach to toxicology could save time, money, and animal resources. Some methods and kits for determining toxicity have been disclosed. See, for example, WO 00/47761, WO 01/32928, U.S. Patent Nos. 5,585,232; 5,589,337; and 5,811,231; and pending U.S. provisional patent applications 60/220,057, 60/254,232, 60/264,933, and 60/308,161.

[0004] With the advent of molecular and recombinant technology, genomic and molecular analysis provides another method by which toxicity may be measured. Differential gene expression technology involves detecting the change in gene expression of cells exposed to various stimuli. The stimulus can be in the form of growth factors, receptor-ligand binding, transcription factors, or exogenous factors such as environmental agents, chemicals, or pharmaceutical compounds.

[0005] Several methods are available for detecting differential gene expression. One method is using an array of polynucleotides. A polynucleotide microarray may include genes for which full-length cDNAs have been accurately sequenced and genes which may be defined by high-throughput, single-pass sequencing of random cDNA clones to generate expressed sequence tags (ESTs). Researchers focused on detecting changes in expression of individual mRNAs can use different methods to detect changes in gene expression e.g., microarray, gel electrophoresis, etc. Other methods have focused on using the polymerase chain reaction (PCR) and/or reverse transcriptase polymerase chain reaction (RT-PCR) to define tags and to attempt to detect differentially expressed genes. Many groups have used PCR methods to establish databases of mRNA sequence tags which could conceivably be used to compare gene expression among different tissues. See, for example, Williams, J. G. K., Nucl. Acids Res. 18:6531, 1990; Welsh, J., et al. Nucl. Acids Res., 18:7213, 1990; Woodward, S. R., Mamm. Genome, 3:73, 1992; and Nadeau, J. H., Mamm. Genome 3:55, 1992. This method has also been adapted to compare mRNA populations in a process called mRNA differential display.

[0006] The process of isolating mRNA from cells or tissues exposed to a stimulus, e.g., drugs or chemicals, and analyzing the expression with gel electrophoresis can be laborious and tedious. To that end, microarray technology provides a faster and more efficient method of detecting differential gene expression. Differential gene expression analysis by microarrays involves nucleotides immobilized on a substrate whereby nucleotides from cells which have been exposed to a stimulus can be contacted with the immobilized nucleotides to generate a hybridization pattern. This microarray technology has been used for detecting secretion and membrane-associated gene products, collecting pharmacological information about cancer, stage specific gene expression in Plasmodium falciparum malaria, translation products in eukaryotes, air-pollutant-induced lung injury, and a number of other scientific inquiries. See, for example, Diehn M, et al., Nat. Genet. 25(1): 58-62 (1993); Scherf, U., et al., Nat Genet. 24(3): 236-44 (1993); Hayward R.E., et al., Mol. Microbiol. 35(1): 6- 14 (1993); Johannes G., et al., Proc. Natl. Acad. Sci. 96(23): 13118-23 (1993); andΝadadur, S.S., et al, Mud. Toxicol. 12(12): 1239-1254 (2000). Microarray technology has also been used in exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis and in rats. See, for example, Wilson M., et al. Proc Natl Acad Sci. 96(22): 12833-8 (1999); Waring, J.F., Toxicol. Lett. 120(l-3):359-368 (2001); Cunningham, M.J., Ann. NY. Acad. Sci. 919:52-67 (2000); and Bulera, S.J., Hepatology 33(5): 1239-1258 (2001). [0007] There exists a need for methods that are fast, efficient, cost-effective, capable of generating large amounts of toxicology data, and could spare many animals from being the subjects of laboratory tests. There also exist a need for a method of effectively selecting genes which are toxicologically relevant to agents being tested and can predict toxicity on a cellular, organ, or system level. There also exists a need for a toxicological database of information whereby one can obtain information about one or more agents being tested and how that agent(s) affects a particular organ or system and algorithms which may be used to identify toxicologically relevant genes and correlate toxicity between agents and target genes.

[0008] Molecular toxicology analysis or toxicogenomics can provide a vast amount of information in the form of a database from a collection of toxicological response data that would be useful in toxicological analysis. The invention and its embodiments provided herein fulfill the aforementioned needs.

SUMMARY OF THE INVENTION

[0009] Disclosed herein are methods of identifying and isolating human genes which are toxicologically relevant and methods of using these toxicologically relevant human genes to determine toxic responses to an agent. Further, arrays containing the human genes, methods of making these arrays, and methods of using these arrays are provided. Also disclosed herein are primer sequences for toxicologically relevant rat genes which are useful for obtaining the toxicologically relevant human homologues.

[0010] In one aspect, a method of identifying a toxicologically relevant human gene is disclosed whereby the gene expression profile of untreated human cells is obtained as well as a gene expression profile of human cells treated with an agent. The gene expression profile of untreated human cells is compared with the gene expression profile ofthe treated human cells to obtain a gene expression profile indicative of a toxicological response. In some aspects, human cells can be any type of cells including but not limited to biological samples from liver, lung, heart, kidney, spleen, testes, thymus, brain, cultured primary human cells, or cells lines obtained from commercial or other sources (e.g., ATCC). The agent can be any type of synthetic or non-synthetic compound including but not limited to agents listed in Table 3.

[0011] In another aspect, a method of isolating human genes indicative of a toxicological response to an agent is provided wherein sequences of mammalian, non-human genes associated with toxicological responses are provided, primers for human genes homologous to said mammalian, non-human genes associated with toxicological responses are provided; and the primers are used to amplify human gene sequences from human cDNA libraries. [0012] In yet another aspect, a method for determining a toxicological response to an agent is provided wherein cells are exposed to an agent and a first gene expression profile is obtained and then compared to a gene expression profile of toxicologically relevant human genes to determine if the first gene expression profile is indicative of a toxicological response. In one aspect, the gene expression profiles of one or more toxicologically relevant human gene(s) are stored in a database. In another aspect, a database containing multiple gene expression profiles of toxicologically relevant human genes is used. [0013] In yet another aspect, a method for determining a toxicological response to an agent in an organ is provided wherein cells from the organ are exposed to an agent and a gene expression profile is obtained and then compared to a gene expression profile of toxicologically relevant human genes to determine if the first gene expression profile is indicative of a toxicological response in an organ.

[0014] In another aspect, a method for screening an agent (e.g. , drug, medicament, or pharmaceutical composition) for potential toxicological responses is provided wherein cells are exposed to an agent; and a gene expression profile is obtained and then compared to a gene expression profile of toxicologically relevant human genes to determine if the first gene expression profile is indicative of a toxicological response in genes associated with toxicological responses. In one aspect, a database containing at least one gene expression profile of toxicologically relevant human genes is used for comparison. [0015] In one aspect, the invention relates to methods of identifying human genes and gene sequences which are indicative of a toxicological response. These genes and their gene expression profiles are stored in a database. The database is useful for toxicological studies and analysis, particular when applied to the screening, development, and testing of potential new drugs. A panel of genes indicative of toxicity can vary between organs different in time of exposure to one or more agents, resulting effects of agent(s) and, different compounds. [0016] In another aspect, a method for generating a human array comprising at least ten human genes which are indicative of a toxicological response is provided. Genes indicative of toxicological response are immobilized to a substrate.

[0017] In another aspect, an array is provided comprising at least ten human toxicological response genes or a portion thereof immobilized on a substrate. The human genes are assembled in an array such that at least 2 genes, more preferably at least 5 genes, more preferably at least 10 genes, more preferably at least 20 genes, more preferably at least 30 genes, even more preferably at least 40 genes, more preferably at least 50 genes, more preferably at least 100 genes, more preferably at least 250 genes, more preferably at least 350 genes, more preferably at least 400 genes, more preferably at least 500 genes, more preferably at least 600 genes, more preferably at least 750 genes, more preferably at least 850 genes, and more preferably at least 1000 genes are assembled on such array. In one aspect, the toxicologically relevant genes are attached to the array substrate by covalent linkage. In another aspect, the genes or portions thereof are capable of hybridization to expressed nucleic acids derived from a cell and are capable of indicating a toxicological response ofthe cell to said agent. [0018] In yet another aspect, a method for obtaining a gene expression profile is provided whereby a population of cells is exposed to an agent, cDNA from the population of cells is obtained, labeled, and contacted with the array comprising toxicologically relevant genes. [0019] In yet another aspect, a method for obtaining a human homologue of a toxicologically relevant non-human gene is provided whereby the sequence of a human homologue is obtained by using the sequence of said non-human gene in a sequence search; primers to the human homologue are provided; and primers to the human homologue are used to amplify a sequence ofthe human homologue from a human cDNA library.

[0020] In still another aspect ofthe invention, primer sequences that are used for identifying human genes are disclosed. These primer sequences can be used for probes, for PCR-related amplification, included on an array chip for identifying nucleotide sequences related to toxicological responses, or for identifying and isolating novel human genes. Sequences of such primers and methods of using thereof are disclosed herein and in Table 2. [0021] In yet another aspect, toxicologically relevant human sequences are cloned and/or maintained in expression or cloning vectors. [0022] In yet another aspect, expression or cloning vectors comprising human toxicologically relevant genes are maintained in suitable host cells. [0023] A method for determining a toxicological response to an agent is provided, the method comprising: (a) exposing cells to an agent or obtaining cells derived from an individual exposed to an agent; (b) obtaining a test expression profile of one or more human toxic response genes in the cells, such as the genes identified in and corresponding to the full or partial gene sequences disclosed in the Tables herein, such as Table 1, 2 and 5; and (c) comparing the test expression profile to a reference gene expression profile of human toxic response genes indicative of toxicity, thereby to determine the presence of a toxic response to the agent. The cells may be derived, for example, from the liver, lung, heart, kidney, spleen, testes, thymus, skin, bone, muscle, gastrointestinal tract, skin, bone, blood, or brain, thyroid, muscle, nucleated cells ofthe blood, gastrointestinal tract or pancreas. Such cells may optionally be cultured cells. The cells may be from an organ or body fluid such as blood or cells in culture, and the test expression profile of human toxic response genes can be compared to the reference gene expression profile, to determine the presence of a toxicological response in the organ.

[0024] The cells in which a toxicological response is determined can be human. The cells may also may be primate, such as primates closely related to human.

[0025] The gene expression profile may be obtained by measuring RNA or protein levels. RNA levels may be measured by hydridization to an array, or other methods, such as real-time polymerase chain reaction, Rnase protection, Northern blot, electrochemical hybridization detection, or branched-chain, to quantitatively detect levels, for example, of messenger RNA. [0026] The toxicity ofthe agent may be evaluated by determining if there is a significant correlation between the test expression profile and the reference expression profile. This correlation can be formally determined by a number of statistical correlation measures using computer assisted statistical analysis methods available in the art or other methods disclosed herein. An observed correlation can indicate that the agent has a similar expression profile to other agents in a database with the inference that the agent will have similar toxic properties. In addition to matching expression profiles for agents, the agent may correlate with expression profiles that are indicative of a specific toxic endpoint. This would allow determination of specific toxic properties. The toxicity ofthe agent may also be evaluated by examining the profiles for expression specific marker genes using models that have been shown through analysis of gene expression databases to be predictive of specific toxicity endpoints. Methods which may be used in the practice ofthe invention, and examples of identification and use of predictive markers are described in U.S. Provisional Appl. Nos. 60/313,080, 60/361,128 and 60/379,861. The reference gene expression profiles may be profiles obtained by previously exposing cells to a toxic agent or profiles obtained from cells of individuals previously exposed to a toxic agent. The reference gene expression profiles indicative of toxicity are, for example, stored in a database and will consist of expressions that can be categorized by agents as well as expressions that can be categorized by specific toxic endpoints. The reference gene expression profiles may be, for example, profiles obtained by previously exposing cells to a toxic agent at various doses and for various amounts of time or by other methods disclosed herein. [0027] In one embodiment, the agent is a pharmaceutical composition such as a drug or diagnostic agent, and the method comprises a method of screening the agent to determine a toxicological response ofthe pharmaceutical agent in the cells. In one embodiment, rapid screening of multiple agents and multiple tissues can be implemented. This can be performed using automated or semi-automated equipment for high-throughput exposure of cells to multiple agents, processing of exposed cells and analysis of gene expression. [0028] The cells exposed to the agent may be cells obtained from a human tissue or body fluid sample, or cultured cells, which are, for example, exposed in vitro to the agent, or the cells may be from a human subject who was exposed to a pharmaceutical or industrial agent. The agent may be exposed to the cells at various concentrations or for various amounts of time or by various routes of exposure. The test expression profile of at least two human toxic response genes in the cells is obtained, or, for example, at least 10, at least 20, at least 50, at least 200 or at least 500 human toxic response genes. [0029] In another embodiment, an array comprising one or more polynucleotides that are the genes corresponding to the full or partial gene sequences disclosed herein, for example, in Tables 1, 2, or 5, or fragments of at least 20 nucleotides thereof, or fragments that are, or are at least, 30, 40, 50, 100, 200, 300, 400, 500 or 600 nucleotides long. The genes may be responsive, e.g., in kidney, liver, spleen, heart, brain, lung, testis thymus, blood, skin or brain cells. The array may include, e.g., at least 25, 50, 200, 500 or more ofthe polynucleotides.

[0030] In another embodiment, gene expression can be measured by any of a variety of methodologies for quantitative detection of specific RNA species coded for by the genes corresponding to the sequences herein, for example, in Tables 1, 2 or 5. These methods include real-time polymerase chain reaction, RNase protection, Northern blot, electrochemical hybridization detection, branched-chain or other methods to quantitatively detect levels of messenger RNA. The expression may be measured, for example, for at least 1, 2, 10, 20, 50, 100, 200, 300, 400, or 500 genes.

[0031] In another embodiment, gene expression can by measured by any of a variety of methodologies for quantitative detection of specific protein species coded for by genes corresponding to sequences herein including those identified in Tables 1, 2 and 5. These methods would include use of specific antibodies in formats such as enzyme-linked immunoabsorbent assays, Western blots and mass- spectrometry methods.

[0032] The disclosure of all patents, patent applications, provisional applications and publications referred to herein are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE TABLES [0033] Table 1 is a list of toxicologically relevant human sequences and primers which may be used to obtain the toxicologically relevant human sequences.

[0034] Table 2 is a chart listing toxicologically relevant rat genes, primers which can be used for obtaining toxicologically relevant rat genes, and primers which were used to isolate human toxicologically relevant genes which are homologues of toxicologically relevant rat genes.

[0035] Table 3 is a list of agents which can be or are used in obtaining toxicologically relevant human genes.

[0036] Table 4 is a chart with human microarray data generated after exposure of human hepatocytes, in vitro, to 3 OmM amiodarone, 50mM chlorpromazine, lOmM paracetamol, 15mM perhexiline or 50mM tacrine. All genes were repressed or induced at least 2-fold. Some ofthe genes that are up- or downregulated are known to be toxicologically relevant and others are not generally known to be toxicologically relevant.

[0037] Table 5 is a list oftarget sequences obtained from human gene sequences cloned using rat sequence-derived primers listed in Table 2 DETAILED DESCRIPTION OF THE INVENTION

[0038] Throughout this disclosure, various publications, patents, patent applications, published patent specifications, and other references are indicated by an identifying citation. All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.

General Techniques

[0039] The practice ofthe present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill ofthe art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Handbook of Experimental Immunology (D.M. Weir & CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); The Immunoassay Handbook (David Wild, ed., Stockton Press NY, 1994); Antibodies: A Laboratory Manual (Harlow et al., eds., 1987); Methods of Immunological Analysis (R. Masseyeff, W.H. Albert, andN.A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993); Principals and Methods in Toxicology (A. Wallace Hayes, ed., 2000); Analytical Methods in Toxicology (H.M. Stahr, 1991); and PCR Protocols in Molecular Toxicology (John P. Nanden Heuvel, ed., 1997).

Definitions

[0040] "Toxicity", as used herein, refers to the microscopic or macroscopic responses of cells, tissues, organs or systems to low, average, or high doses of an agent. Toxicity often results in toxic side effects that are different, in either degree or kind, from the response ofthe majority of patients at the recommended dose of a pharmaceutical compound. Manifestations of toxicity can include but are not limited to climcal symptoms (e.g., dizziness or nausea), abnormal serum chemistry, hematology or urinalysis values, changes detectable as histopathology results, or abnormal gross appearance ofthe tissues and organs at necropsy. [0041] A "toxicological response" as used herein refers to a cellular, tissue, organ, or system level response to exposure to an agent and includes, but is not limited to, the differential expression of genes and/or proteins encompassing both the up- and down-regulation of expression of such genes; the up- or down- regulation of genes which encode proteins associated with the repair or regulation of cell damage; or the regulation of genes which respond to the presence of an agent.

[0042] The terms "toxicity gene(s)", "toxicologically relevant gene(s)", and "toxic response gene(s)" as used herein are interchangeable. These terms can be defined as a gene whose messenger RNA or protein level is altered by an agent (e.g., an adverse stimuli). The specific set of genes that are induced in cells is dependent upon, inter alia, the type of damage or toxic threat caused by the agent and which organs are most threatened. In addition to the up-regulation or down- regulation of genes which respond to specific toxic threat, genes which encode functions not appropriate under conditions of toxic injury may be down-regulated. [0043] As used herein, the term "gene" refers to polynucleotide sequences which encode protein products and can encompass RNA, mRNA, cDNA, single stranded DNA, double stranded DNA, and fragments thereof. Genes can include introns and exons.

[0044] The term "gene sequence(s)" refers to gene(s), full-length genes or any portion thereof.

[0045] "Gene expression indicative of toxicological response" as used herein refers to the relative levels of expression of a toxicity gene or toxic response gene. Profiles of gene expression profiles may be measured in a sample, such as samples comprising a variety of cell types, different tissues, different organs, or fluids (e.g., blood, urine, spinal fluid, sweat, saliva, or serum). [0046] As used herein, the term "agent" means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, or antibody fragment. Physical agents, such as radiation, is also encompasses in this definition. Various compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "agent". In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Agents can be tested and/or used singly or in combination with one another. An "agent" to which an individual has a toxicological response can also be any substance to which an individual exhibits a toxicological response and includes, but is not limited to, drugs, pharmaceutical compounds, household chemicals, industrial chemicals, environmental chemicals, and other chemicals and compounds to which individuals may be exposed. Exposure to an agent can constitute physical contact as well as secondary contact, such as inhalation and environmental exposure. As used herein, "agent" and "compound" may be used interchangeably.

[0047] As used herein, "array" and "microarray" are interchangeable and refer to an arrangement of a collection of nucleotide sequences in a centralized location. Arrays can be on a solid substrate, such as a glass slide, or on a semi- solid substrate, such as nitrocellulose membrane. The nucleotide sequences can be DNA, RNA, or any permutations thereof. The nucleotide sequences can also be partial sequences from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences.

[0048] "Hybridization" or "hybridize" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases ofthe nucleotide residues. The hydrogen bonding is sequence-specific, and typically occurs by Watson-Crick base pairing. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0049] Hybridization reactions can be performed under conditions of different "stringency". Relevant conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as formamide, and the washing procedure. Higher stringency conditions are those conditions, such as higher temperature and lower sodium ion concentration, which require higher minimum complementarity between hybridizing elements for a stable hybridization complex to form. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art: see, for example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, Fritsch & Maniatis, 1989). When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, those polynucleotides are described as "complementary". A double-stranded polynucleotide can be "complementary" to another polynucleotide, if hybridization can occur between one ofthe strands ofthe first polynucleotide and the second. Complementarity (the degree that one polynucleotide is complementary with another) is quantifiable in terms ofthe proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.

[0050] An "individual" is a vertebrate, preferably a mammal, for example a human. Mammals include, but are not limited to, humans, farm animals, sport animals, pets, primates, mice, and rats.

[0051] The term "sample" or "biological sample", as used herein, refers to substances supplied by an individual. The samples may comprise cells, tissue, parts of tissues, organs, parts of organs, or fluids (e.g., blood, urine, sweat, saliva, or serum). Samples include, but are not limited to, those of eukaryotic, mammalian or human origin.

[0052] The terms "protein", "polypeptide", and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. It also may be modified naturally or by intervention; for example, disulfide bond formation, glycosylation, myristylation, acetylation, alkylation, phosphorylation or dephosphorylation. Also included within the definition are polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids) as well as other modifications known in the art.

Methods of the Invention

[0053] Human genes which are toxicologically relevant have been identified and are disclosed herein. Methods of identifying toxicologically relevant human genes or genes that are likely to be toxicologically relevant are described herein. In addition, methods of isolating and using toxicologically relevant genes are disclosed. In one embodiment, toxicologically relevant genes are used to make arrays. The arrays can be used for drug screening purposes to determine toxicological response to an agent.

Identifying a set of toxicologically relevant genes

[0054] Identification of a set of toxicologically relevant genes can be achieved by several methods. One method which can be used is to clone genes previously described to be relevant in toxicology or to clone genes putatively identified to be important for a toxicological response because ofthe known or suspected function ofthe gene or because ofthe functional relationship of that gene to other genes which play a role in toxicological responses. Using published sequences, for example in literature or from GenBank, primers can be made and then used to PCR amplify from a relevant library to obtain the toxicologically relevant gene of interest which can then be cloned into a plasmid or an expression vector, depending on the use desired. The gene can be placed amongst other toxicologically relevant genes in a microarray for high-throughput testing, as disclosed infra. [0055] Alternatively, for replication to high copy numbers, a plasmid may be used to grow high copies ofthe toxicologically relevant gene of interest which can then be purified by any commercially available kit (e.g., from Qiagen or Promega). The purified toxicologically relevant gene may be used for "spotting" in a microarray or alternatively, the purified nucleic acid can then be inserted into an expression vector, transfected into mammalian cells, e.g., human cells, and then the cells can be exposed to a compound and observed for toxicological responses. Toxicity may be ascertained by any number of methods known to one of skill in the art such as observing changes in cell morphology or re-arrangement of cytoskeleton, which can be determined by examination under a microscope, or alternatively, cell apoptosis or necrosis, or biochemical changes such as leakage of enzymes or ions from the cell. In another alternative, "transcriptome profiling", described in greater detail below, may be used whereby nucleic acid can be isolated from both the exposed and unexposed cells and examined to determine which level ofthe compound causes the up-regulation or down- regulation ofthe toxicologically relevant gene of interest. [0056] Another method which can be used to identify a set of toxicologically relevant genes is to test available human genes for the genes' response using tissues from human toxicity studies and select those with differential expression. Differential expression may be assessed by any number of methods. One method which may be used is by microarray analysis. Provided herein are methods of using microarray analysis to determine differential gene expression. Another method of determining differential gene expression is by reverse transcriptase- polymerase chain reaction (RT-PCR), e.g., Taqman® technology (Foster City, CA). Yet another method which could be used to detect differential gene expression is Invader® technology, commercially available from Third Wave (Madison, WI). Yet another method which may be used to determine differential expression is Northern blot analysis.

[0057] Other methods which may be used include open systems such as cDNA-AFLP and SAGE (Klein, P.E., et al. Genome Res. 10(6):789-807 (2000); Wang, X. and Feuerstein, G.Z., Cardiovasc Res. 35(3):414-21 (1997)) Feuerstein, G.Z. and Wang X. Can J. Physiol Pharmacol. 75(6):731-4 (1997); Hough, CD. et al., Cancer Res. 60(22):6281-7 (2000); Ye, S.Q., et al., Anal Biochem. 287(1): 144-52 (2000)). An "open system" allows the entire transcriptome to be analyzed instead of a defined set of genes.

[0058] Alternatively, comparisons between gene expression profiles from control human cells (or human cell lines) and human cells (or human cell lines) treated with an agent can be used to select responsive genes. This is referred to herein as "transcriptome profiling". This method empirically determines which genes are toxicologically relevant by analyzing differential gene expression. In this embodiment, experimental human cells are divided into two groups. One group is exposed to one agent at different concentrations for different lengths of time. Another group of human cells are not exposed to any agent and serve as the control group. Once the experimental group is exposed to at least one agent, then RNA of both groups is isolated and reverse transcribed in PCR reactions to generate cDNA which in turn is amplified to generate double stranded DNA. The PCR is performed in the presence of a radioactive DNA substrate that is incorporated into the double stranded DNA. On a polyacrylamide gel, the DNA derived from the treated cells is separated by length next to the DNA derived from untreated population. The intensity ofthe resulting band or bands is compared between the treated and untreated groups of cells. Bands that show different radioactive intensity are excised from the gel, amplified by PCR, cloned, and sequenced. The sequences are compared with known gene sequences in the public databases such as GenBank. In this manner, novel human genes, in addition to known human genes with varying degrees of similarity, which are toxicologically relevant can be discovered and identified. [0059] If a partial sequence of a novel human gene is discovered, the technology, texts (see Sambrook et al. infra), and resources available to a skilled artisan would enable the sequencing ofthe of remainder ofthe gene and obtain a full-length gene without undue experimentation. One method of obtaining the remaining portion of a novel human gene is to make primers corresponding to the part ofthe novel human gene which are known combined with random primers and then use the primers in PCR reactions with a human cDNA library. The PCR reaction are run on a standard agarose gel and amplified bands are identified, excised from the gel, and sequenced.

[0060] Yet another method which may be utilized to identify a set of 'toxicologically relevant genes is by obtaining human homologues to toxicologically genes of other species (e.g., rat). Methods for identifying and obtaining toxicologically relevant rat genes are disclosed in pending U.S. applications 60/264,933 and 60/308,161. Primers may be made from toxicologically relevant genes from non-human individuals and used in PCR reactions with human cDNA libraries to obtain a human homologue of a non- human toxicologically relevant gene.

[0061] In another alternative, sequences of human homologues of a toxicologically relevant non-human (e.g., rat) gene may be obtained by using the sequence of non-human gene in a sequence search (e.g., a BLAST search) to find known human sequences which have high homology to the non-human (e.g., rat) gene. Primers to the human homologue may be synthesized and then used to amplify a sequence ofthe human homologue from a human cDNA library. Examples of primers which may be used are disclosed in Table 2 and the protocols which have been used are disclosed in Examples 1-5 and 9. Successfully cloned human gene sequences using primers described in Table 2 are presented in Table 5. Methods of this embodiment are further detailed in the Examples section.

[0062] Each of these methods is disclosed in greater detail below. Other factors to consider in identifying toxicologically relevant genes include, but are not limited to, selection of one or more agent(s), the dosage amount to administer, and routes of administration.

Selection of agent(s)

[0063] The agent to be tested can be selected on the basis of different criteria. One method of selecting which compound to test is damage observed in specific organs. For example, cisplatin, amphotericin B and gentamicin have been observed to cause kidney tubular epithelial cell damage. Another example, liver peroxisome proliferation has been observed to be affected by clofibrate, gemfibrozil, and WY 14,643. Another basis for selection is molecular and biochemical action. For example, cisplatin causes apoptosis and reactive oxygen species, amphotericin B causes increased permeability of cell membranes to ions and renal vasoconstriction, and gentamicin causes phospholipid accumulation in lysosomes.

[0064] Other toxicants affect an organ in general, for example, some kidney toxicants include but are not limited to cisplatin, gentamicin, puromycin, and amphotericin B. Liver toxicant include but are not limited to chlorpromazine, clofibrate, diflunisal, tetracycline, erythromycin, and ethanol. Immunotoxicants include but are not limited to cyclosporin A, lipopolysaccharide (LPS), hydroxyurea, phenylhydrazine, dexamethasone, estradiol, and tamoxifen. Heart toxicant includes but is not limited to doxorubicin. Multiorgan toxicants include but are not limited to methotrexate and cadmium chloride. [0065] Another criterion for selecting an agent is based on exposure to the agent, for example, those agents to which an individual might be exposed to on a regular basis, either in the environment (e.g., occupational exposure, accidental exposure, or voluntary exposure), by prescription, or over-the-counter drug can be selected for testing. Another criterion for selection of an agent is regulatory approval. For example, those agents which are required to be tested for toxicity for FDA-approval or alternatively for other toxicity requirements, for example in pre-clinical or clinical trials can be selected. Table 3 lists some agents which may be selected given the criteria above.

Determination of dosage

[0066] Dosages to use in experiments with human cells or biological/clinical samples can be determined using several methods. One method is to use reported dosages (e.g., obtained in pre-clinical or clinical studies or published in clinical reports) as a starting point and dose incrementally above and below the reported dosage. Increments can be at least ±1%, 5%, 10%, 25%, 35%, 45%, 50%, 60%, 70%, 80%, 90%, or 95%. Alternatively, dosages which are known to affect non- human individuals (e.g., rats, primates, dogs, etc.) which have similar gene expression profiles may also be used as a starting point and then dosages may be incrementally increased or decreased. If non-human individuals (e.g., rats, primates, dogs, etc.) are to be used for comparison purposes, the upregulation or downregulation of markers in the blood including but not limited to serum chemistry values and hematology values can be used to determine if toxicity has been reached. Alternatively, examining the histopathology of organs, in particular, organs which are the specific targets ofthe agent of interest, may be used to determine if a pathological change has occurred in response to administration ofthe agent. Another method which may be used is to determine the molecular changes by analyzing the gene expression in response to administration of different doses of a agent by the methods disclosed infra. [0067] Determination ofthe dosage experimentally using cell cultures is affected by many factors: the nature ofthe agent, its potency, mechanism of action, type of cell which is the target ofthe agent, and number of cells. To determine the dosage required experimentally, a low dosage level ofthe agent is added and then in a step- wise manner, the dosage is increased as well as length of time exposed to the agent. If the agent is lipophilic and easily crosses the lipid bilayer of cells, a lower initial concentration may be used and/or shorter length of time exposed to the agent. If the agent possesses a nature that would not cross the cell barrier easily and would need to be actively or passively transported across cell membranes, then a slighter higher initial concentration may be used and/or longer length of time exposed to the agent. Increasing dosage step- wise while monitoring toxicological response and morphology ofthe cells, rate of death of the cells, and growth patterns allows the skilled artisan to determine the dosage at which a toxicological response occurs. Toxicological responses may occur which are visible changes, including but not limited to, physical structure and integrity of the cells (e.g., morphology, growth pattern, etc.). Monitoring for cellular toxic responses as well as molecular toxic responses, e.g., differential gene expression increases the likelihood of finding preferable dosages. [0068] Changes in gene expression may be toxicologically significant. The point at which toxicologically relevant gene expression becomes even more relevant is at that dosage at which removal or diminishment ofthe treatment no longer results in a return to normalcy, e.g.,, the state of a cell, organ, or system that existed prior to the treatment with the agent. Treatments beyond a certain dosages or time period may commit the cell to a toxicologically-relevant fate. This toxic dosage will be reflected by an identifiable gene expression pattern, which will be distinct from the pattern observed below the toxic dosage. [0069] Dosage response is an important concept in toxicology. Depending on the dosage of a toxin or agent which may be toxic, the gene expression profile of a particular gene may vary. One way that this can be envisioned is by observing the changes in fold induction of a particular gene when analyzed using the arrays of this invention. The dosages determined in dose response curves may be useful in determining "threshold" levels of toxicity, for example for FDA approval. Methods of analyzing gene expression and how to correlate gene expression data are provided herein.

Administration of an agent

[0070] If non-human individuals (e.g., rats) are to be used for comparison purposes, administration of an agent to the non-human individual may be achieved by various routes. It will be readily appreciated by those skilled in the art that the route can vary, and can be intraperitoneal, intravenous, subcutaneous, transcutaneous, intramuscular, enteral, transdermal, transmucous, sustained release polymer compositions (e.g., a lactide polymer or co-polymer microparticle or implant), perfusion, pulmonary (e.g., inhalation), nasal, oral, etc. Injectables can be prepared in conventional forms, either as liquid solutions or suspension, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, aqueous dextrose, glycerol, ethanol or the like. Formulations for parenteral and nonparenteral drug delivery are known in the art and are set forth in Remington's Pharmaceutical Sciences,' 18th Edition, Mack Publishing (1990). [0071] The carrier must be acceptable in the sense of being compatible with the agent to be tested and not deleterious (e.g., , harmful) to the human to be treated. The composition or formulation to be administered can contain a quantity ofthe agent in an amount sufficient to effect one or more toxicological response in the human, either on a molecular level or on a physiological level.

Method of identifying toxicologically relevant genes using known human genes

[0072] In one embodiment, sequences of human genes which are toxicologically relevant are known, either in the art or in a publicly available database, e.g., GenBank. The first method that is used to identify human genes involves searching a public database, for example GenBank, for human genes already known to have a toxicological response. Using the known toxicologically relevant human sequences, primers are designed and used in PCR reaction to amplify the human gene sequences from a cDNA library. The cDNA library can be made from different human cells. The generation of a cDNA involves reverse transcribing isolated RNA and is well-known in the art (see for example, Sambrook et al. supra). The human gene fragments, amplified by PCR, are cloned into any standard plasmid expression vector which can be obtained from numerous commercial sources (e.g., Promega, InNifrogen, New England BioLabs, etc.) and sequenced. The resulting sequence information is then compared to the GenBank database to confirm that the cloned DNA is the specific human gene for which the primers were designed. Upon positive confirmation ofthe sequence, the amplified gene sequence is then added to the panel of genes to be included in the array. Methods of including toxicologically relevant genes are disclosed infra. [0073] In another embodiment, human genes which are toxicologically relevant are known. This method to identify human genes utilizes known sequences of toxicologically relevant non-human genes (e.g., rat genes identified in pending U.S. applications 60/264,933 and 60/308,161 or canine genes identified in pending U.S. application 60/227,057 and the U.S. application claiming priority thereto). These toxicologically relevant non-human genes may be from a non-human species including, but not limited to rats, primates, and other mammals. Human homologues ofthe non-human genes can be identified through query of human sequence databases such as GenBank with comparative sequence analysis programs such as the Basic Local Alignment and Search Tool (Altschul, S.F., et al., Nucleic Acids Res. 25:3389-3402 (1997)). Primers to these toxicologically relevant non-human genes or identified human homologues are designed, synthesized, and are subsequently used in PCR reaction with human cDNA libraries to amplify the homologous human gene. If non-human genes were used to design the primers, the homologous human gene may or may not be the exact sequence as the non-human gene with which the primers were designed. Amplified human gene is then added to the panel of genes to be included in the array.

[0074] In yet another embodiment, target sequences for inclusion in a human array are obtained by de novo synthesis of polynucleotides. The polynucleotide may be synthesized directly on the slide or may be synthesized by other means and then attached to the slide. The target sequences are from genes which can indicate one or more toxicological responses.

Transcriptome profiling

[0075] Transcriptome profiling is a generic term that can be applied to measurement of a large a variety of transcripts. Various methods can be used for the profiling such as microarray, PCR, and differential display, SAGE, Invader®, etc. Several methods of differential display can be used to identify genes of interest. Differential gene expression can be observed by using techniques involving gel electrophoresis and polynucleotide microarrays or commercially available technologies, e.g., Invader® or Taqman®.

[0076] In one method, the results of PCR synthesis of mRNA (converted to cDNA before PCR) isolated from tissues of treated and control human cell lines are subjected to gel electrophoresis, and the bands produced by these mRNA populations are compared. Bands present on an image of one gel from one mRNA population, and not present or present with much less intensity on another, correspond to the presence of a particular mRNA in one population and absent or at much lower levels in the other, and thus indicate a gene that is likely to be differentially expressed. Messenger RNA derived from control and treated human or cell lines can be compared by using arbitrary oligonucleotide sequences (random 13-mers) as a 5' primer and a set of 3 oligonucleotides complimentary to the poly A tail as a 3' fluorescent labeled "anchor primer". These primers are then used to amplify partial sequences of mRNAs with the addition of deoxyribonucleotides. These amplified sequences are then resolved on a sequencing gel. The sequencing gels are then compared to each other to determine which amplified segments are expressed differentially. See, for example, Liang, P. et al. Science 257:967, 1992; Welsh, J. et al., Nucl. Acid Res. 20:4965, 1992; and Liang, P., et al., Nucleic Acids Res. 21(14):3269-75 (1993)). [0077] An open system may be used whereby human cells are exposed to drugs and/or chemicals at different concentration and then harvested at different time points. Human cells can be obtained from various sources including, but are not limited to, tissue samples, organs, blood, skin, biological fluids (e.g., urine, spinal fluid, semen, etc.), and cell lines. Transcriptome profiling can also be done using tissues, biological fluids, etc. from humans who have been dosed in vivo during clinical trials or during one or more clinical treatment(s). Immortalized human cell lines may also be used and can be obtained from commercial sources, e.g., Gibco BRL Life Sciences or other sources, e.g., American Type Culture Collection (ATCC). Other methods of obtaining human cells include isolating cells obtained from tissue biopsies, blood, skin, or biological fluids, for example from humans dosed in vivo. In an alternative, non-human individuals (e.g., rats) may be used for comparison, as disclosed herein. Tissue samples, cells, or cell lines from a non-human individual may be utilized as well for transcriptome profiling.

[0078] As is well known to one of skill in the art, isolating cells from tissue samples can be achieved using any variety of techniques. In obtaining tissue samples, for example during necropsy, it is important to avoid conditions that would cause degradation of nucleic acids (e.g., RNA). [0079] One method which may be used is to digest a tissue sample in an enzymatic solution to break up connective tissue and then agitate cells in the digested tissue to separate the cells from the connective tissue. Examples of other enzymes that can be used to digest tissue include neutral proteases, serine proteases including, but not limited to, trypsin, chymotrypsin, elastase, collagenase, and thermolysin. Another method is to homogenize the tissue sample or apply mechanical stress forces to the tissue sample to separate the cells from the basement membranes and allow the cells to become separated from within the tissue. In the alternative, DNA or RNA can be directly isolated from tissue samples, as exemplified in the examples disclosed herein. Isolating cells from blood can be achieved by layering blood over a gradient (e.g., Percoll™ or Ficoll™), spinning the blood-gradient layer in a centrifuge, and extracting the layer of cells from serum.

[0080] Sources from which cells are obtained can be any number of organs, including but not limited to liver, lung, heart, kidney, spleen, thymus, and brain. In one embodiment, liver cells may be used for toxicity studies where the agent to be administered is known or thought to induce liver malfunctions or liver toxicity. In other embodiments, when the target ofthe action delivered by the agent is known, the use of cells deriving from the target organ may yield more beneficial information regarding toxicological responses than if a tissue were selected at random. In another embodiment where the agent to be tested has unknown effects, a panel of cells isolated from different sources may be used. In the alternative, liver cells may be used in the absence of knowledge ofthe agent's target of action because the liver is known to process many toxins. Although the toxicological responses may not be the most ideal compared to the results that one of skill in the art would obtain if the target tissue ofthe agent's action had been used, the benefits of using liver cells would be that toxicologically relevant genes may be identified and then subsequently tested on other organs to determine toxicity in the other organs or alternatively, to identify which organ(s) is the target for the agent. 3/016500

[0081] Human cells obtained ex vivo or from a commercial or other source (e.g., ATCC) can be used fresh or frozen for storage and then cultured in media at time of experimentation. A wide variety of basal cell-sustaining media that can be used to keep the pH ofthe liquid in a range that promotes survival of human cells. Non-limiting examples include F12/DMEM, Ham's F10 (Sigma), CMRL-1066, Minimal essential medium (MEM, Sigma), RPMI- 1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM, Sigma), and Iscove's Modified Eagle's Medium (IMEM). In addition, any ofthe basal nutrient media described in Ham and Wallace Meth. Em., 58:44 (1979), Barnes and Sato Anal. Biochem., 102:255 (1980). Cells can be grown in plates or in flasks and expanded to an amount needed for DNA or RNA isolation. Cells can then be removed from the plate or flask to isolate DNA or RNA. If the cells are adherent, trypsin or another equivalent may be used to release the cells from the plate or flask. Methods of culturing cells and isolating nucleic acids from cells are well-known in the art. [0082] Methods of exposing non-human individuals (e.g., rats) to toxic doses of agents include determination ofthe dosage and routes of administration of agents as described above. Rats may be divided into treated rats that receive a specific concentration ofthe agent and the control rats that only receive the vehicle in which the agent is mixed (e.g., saline). At specified timepoints after administration ofthe agent, a set number of rats (control and treated) may be euthanized by any standard euthanization protocol known in the art and tissues may be collected. The method of collecting the tissues is important and ensures preserving the quality ofthe mRNA in the tissues. Each rat may be heavily sedated with a overdose of CO₂ by inhalation and a maximum amount of blood drawn. Exsanguination ofthe rat by this drawing of blood kills the rat. The body ofthe rat may then be opened up and prosectors may rapidly remove the specified organs/tissues and immediately place them into liquid nitrogen. All ofthe organs/tissues should be completely frozen within 3 minutes ofthe death ofthe rat. The organs/tissues may then be packaged and stored at -80 degrees until needed for isolation ofthe mRNA from a portion ofthe organ/tissue sample. [0083] Nucleotide sequences from tissue samples are isolated using any number of commercially available kits (e.g., from Qiagen, GenHunter®, Promega, etc.). In general, a skilled artisan should take care to keep all reagents, tubes, and instruments sterile as to avoid contaminants which may affect how the results are interpreted. Once DNA or RNA has been isolated from cells which have been exposed to one or more agents, one or more toxicologically relevant genes are identified using the methods described above. The toxicologically relevant genes may be cloned into an expression vector, maintained in an expression vector or alternatively, the expression vector comprising the toxicologically relevant gene sequence may be transformed or transfected into a suitable host cell. Suitable host cells may be obtained from the ATCC or from commercial sources. Methods of isolating toxicologically relevant genes by cloning are further detailed in the Examples.

[0084] In some embodiments, the toxicologically relevant non-human gene may be used to find a homologue in another animal, for example, in humans. The homologue may be then be used as a target or to derive a target for drug development, toxicity screening, prognostic or diagnostic applications (e.g., antigen for antibody development or cellular regulation). [0085] In other embodiments, human genes identified to be toxicologically relevant may be used to generate an array of toxicologically relevant human genes. In this case, the gene may be cloned to facilitate the process of generating an array.

Preparation of Microarray

[0086] The isolated DNA or RNA is amplified to generate a product which can be attached to a substrate. In a preferred embodiment, the substrate is a solid substrate (e.g., a glass slide). The amplification process involves using primers which have a reactive group (e.g., amine group or derivative thereof) on one end ofthe primer, which is incorporated into the amplification product. One example of reactive primers that can be used is Amine Primers from Synthegen (Houston, TX; catalog #5002). The gene fragments which are attached to the glass slide can vary in length. The more nucleotides of a gene that are in the array, the tighter the binding and the greater the specificity in binding can occur. However, it is important to consider that longer fragments are more difficult to amplify and may contain point mutations or other errors associated with amplification. Therefore, the desired length of a gene or a fragment thereof that is to be included in the array should take into consideration the balance between a high specificity of binding obtained with a long (e.g., >1 kb) gene sequence with the high mutational rate associated with a longer fragment.

[0087] The gene fragments attached to the glass slide are at least about 50 base pairs (bp) in length, more preferably at least about 100 bp in length, more preferably at least about 200 bp, even more preferably at least about 300 bp, even more preferably at least about 400 bp, even more preferably at least about 500 bp in length. In one embodiment, the gene fragments are about 500 bp in length. The region of a gene that is used to attach to a solid substrate to generate an array can be any portion ofthe gene, coding, non-coding, 5' end, 3' end, etc. In one embodiment, about 500 base pairs ofthe 3' end of human gene related to toxicological responses are selected to be included in an array. [0088] In some embodiments, the skilled artisan should be aware that the human homologues of toxicologically relevant rat genes to be attached to the array may have different lengths than the toxicologically relevant rat gene. For example, in the instance where the rat gene sequence is longer than the human homologue, the rat gene sequence will have 500 base pairs at the 3' end that the human sequence does not have. In another instance where the human homologue is longer than the rat gene sequence, the human sequence will have 500 base pairs at the 3' end that the rat gene sequence does not have. Either the 3' region can be used or regions of equivalent homology on the gene sequence should be used. [0089] Several techniques are well-known to a skilled artisan for attaching a gene or a fragment thereof to a solid substrate such as a glass slide. One method is to attach an amine group, a derivative of an amine group, another group with a positive charge or another group which is reactive to one end of a primer that is used to amplify a gene or a gene fragment to be included in the array. Subsequent amplification of a PCR product will then incorporate this reactive group onto one end ofthe product. The amplified product is then contacted with a solid substrate, such as a glass slide, which is coated with an aldehyde or another reactive group which will form a covalent link with the reactive group that is on the amplified PCR product and become covalently attached to the glass slide. Other methods using amino propryl silicane surface chemistry and other methods of preparation of microarrays have been disclosed. See, for example, Nuwaysir, E.F., et al. Molecular Carcinogenesis, 24:153-159 (1999); Kane, M.D., et al. Nucleic Acids Res. 28(22):4552-7 (2000); MacBeath G. and Schreiber, S.L., Science 289(5485):1760-1763 (2000); Lockhart, D. J. and Winzeler, E. A., Nature 405(6788):827-836 (2000); Cortese, J.D., The Scientist 14(17):25 (2000); and Cortese, J.D., The Scientist 14(11):26, (2000). Other methods using amino propryl silicane surface chemistry are disclosed by Corning Company (Corning, NY). Products made by Corning Company which include CMT™ Yeast-S288c gene arrays, CMT™ Human Cancer gene arrays, CMT-GAP S™ II coated slides, and CMT™ Hybridization chamber. The website of Corning Company discloses more information about how a skilled artisan may make microarrays. At present, the website may be accessed at <http://www.cmt.corning.com.>. Other methods for making microarrays are disclosed by Haab B.B., et al Genome Biol. 2001 Jan 22; 2(2): RESEARCH 0004.1-0004.13; Sherlock G., et al. Nucleic Acids Res. 2001 Jan 1;29(1): 152-5; and the website of Dr. Pat Brown at Stanford University. At present, the website may be accessed at <http://cmgm.stanford.edu/pbrown>. [0090] In one embodiment ofthe invention, fluorescence-labeled single strand (or "first strand") cDNA probe is made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra. This probe is hybridized to microarray slides spotted with DNA specific for toxicologically relevant genes. Methods for making the array and for labeling and making cDNA probes are disclosed in the Examples. Algorithms for analysis and evaluation of toxicologically relevant genes [0091] A multi-step approach can be used in ranking candidate genes from human genes for possible inclusion on an array. First, three cutoff criteria can be specified for individual gene values from experiments results: 1) Fold Induction/Repression level, 2) Average Fluorescence level ofthe replicate spots (reflection ofthe expression level) and 3) Coefficient of Variation ofthe replicate spots. The initial screening to make the "cut" may be based on expression level and measurement quality.

[0092] Second, gene values that would made the cut can be aggregated into overall scores, and ranked for each gene, may be based on six ranking criteria: 1) Number of slides on which that gene met the cutoff criteria (NC), 2) Percent of consistency between slides (% of time the gene value made the cutoff criteria on the replicate slide for that initial slide) (CC), 3) Average magnitude (absolute value) of fold induction for all occurrences where that gene made the cutoff criteria (FI), 4) Coefficient of Variation of those fold induction scores (unlike all the other ranking criteria, lower is deemed better) (CV), 5) Average fluorescence value of all replicate spots of occurrences where that gene made the cutoff criteria (FL), and 6) Tissue consistency (what percent of cutoff-meeting occurrences of the gene were in the same tissue) (CT).

[0093] Each gene can be assigned a score between 0 and 100 for each ranking criterion. Each ranking criterion score can be computed as follows: The range of values for all genes is computed for the criterion by subtracting the lowest value present among all scores from the highest. The score for each gene is then calculated by subtracting the lowest value present from the value for that gene, then dividing by the range and multiplying by 100. In other words, the score for each gene is the percent above the minimum present toward the maximum. For example, if a gene's score was three-fourths ofthe way between the minimum present and the maximum for that criterion, its score would be 75%. Since for the CV factor (coefficient of variation of fold inductions) lower is deemed better, the score thus computed was subtracted from 100 to invert the percentage. [0094] The final ranking score for each gene can be computed via a weighted combination of its score on the six ranking criteria. If a score could not be computed for a particular criterion, the entire value of that criterion would be removed from the equation, and ranking was based solely on the remaining factors.

Methods of using toxicological response data

[0095] The set of toxicologically relevant human genes and methods of identifying toxicologically relevant genes in human may be used in several embodiments. In one embodiment, toxicity dosages and time of exposure which is required to reach a toxic dose are determined by using the methods disclosed above. In another embodiment, an individual (e.g. , human) can be tested for toxic responses to a particular agent. The individual may be hypersensitive to the agent (e.g., penicillin). Analyzing the individual's gene expression profile may determine if the agent has a toxic effect in the individual. Alternatively, the gene expression profile ofthe individual may be compared with other gene expression profile stored in a database. Methods of using gene expression profiles of toxicologically relevant human genes to generate a useful database is described in further detail below. In another embodiment, the gene expression profiles of toxic responses in non-human species (e.g., rats or mice or other animals) may be determined. This may assist in determining which species is best suited for animal models by assessing which species is most susceptible to toxic responses. In yet another embodiment, the methods and set of toxicologically relevant genes disclosed herein can be used to predict and/or determine drug-drug interaction in an individual. As disclosed supra, gene expression profiles of toxicologically relevant human genes can be compared when dosed with one drug and then compared to a second gene expression profile when dosed with another drug. The toxicologically relevant data may be correlated using the algorithms disclosed herein. The effects of drug-drug interaction may induce a similar set of genes to be up-regulated or down-regulated. The effect may be additive or multiplicative. Alternatively, the effects ofthe drug-drug interaction may induce different sets of genes which are not related in function. In another embodiment, the methods and set of toxicologically relevant genes disclosed herein allow target organs and toxic doses therein to be determined. This is useful in drug design where the drug may have an intended target of one organ but have toxic multi-organ effects. In another embodiment, the methods and set of toxicologically relevant genes may be used to predict toxic response to agents which may take repeated exposure over a period of time for symptoms of toxicity to appear. Examples of such agents are disclosed in Table 3 and can also include one-hit carcinogens (e.g., aflatoxin Bl, dimethylnitrosamine, ENU, etc.) or multi-dose carcinogens (e.g., phenobarbital and WY14,643). The molecular toxic response to these carcinogens may be determined in advance of any macroscopic changes which may occur in response to exposure to these agents.

Method of using toxicological response data to generate a toxicological database and uses thereof

[0096] By collecting many gene expression profiles from certain species, e.g. , humans, in response to one or more agents, a database can be built with a collection of information about toxicological responses. With the database, it could be possible to predict toxicological response to specific agents or combinations thereof. The database can be stored on a computer and in a manner that allow for rapid searching when a comparison is desired. The database could store gene expression profiles for a particular toxin or alternatively, a group of toxins (e.g., kidney-specific toxins). The database could also store gene expression profiles for a group of genes known to be affected by a particular toxin. When a gene expression profile is obtained, it may be compared with the gene expression profiles stored in the database to determine what type of organ is likely to be affected, or alternatively, which genes could also be associated with the toxic response. One or more genes could be analyzed in this manner as well as one or more toxins. The database may be stored in a form that allows for rapid, access and analysis with compatible software programs. [0097] The instant invention of human gene arrays provides an alternative to testing on live animals such as rats, mice, or dogs. The human gene array can provide answers concerning human response to a particular agent by examining the differential gene expression associated with that particular agent using an array or comparison with a human array database. Further, human gene arrays can provide answers about toxicological responses faster and more efficiently than testing in vivo.

[0098] Another use for a database of gene expression profile of toxicologically relevant genes is for comparisons across species. For example, a database comprising human gene expression profiles obtained from in vitro studies can be used to compare to rat gene expression profiles obtained from in vitro studies. If the human in vitro gene expression profile is similar to the rat in vitro gene expression profile in response to a particular agent, then it can be inferred that the rat model would be a good model to use for assessing in vivo responses. The rat in vivo response can be extrapolated to predict human in vivo responses.

[0100] The information generated from using human gene arrays can be used to predict cellular and pathological responses as well as histological changes induced by exposure to agents. This is accomplished by analyzing the differential gene expression observed when human gene arrays are used. Potential drugs or pharmaceutical agents can be tested and data gathered for FDA approval in an accelerated manner and can help pharmaceutical and biotechnology companies generate higher productivity with lower costs in research and development. [0101] The human gene array can also generate information that can be used to predict downstream effects, such as which pathways are affected by certain agents. This is accomplished by looking at the differential gene expression and analyzing which pathways contain the toxicological response genes and also which pathways the genes can affect. This information in turn can be used to predict tissue responses and ultimately whole organ responses. Examples of whole organ responses include but are not limited to organ functions, inflammatory responses, and autoimmune responses. Those of skill in the art can determine when the normal functions of an organ are compromised by exposure to one or more agents which are toxic. For example, a kidney's ability to filter toxins is compromised after an individual has been exposed to an agent. The ability to predict whole organ responses has great potential in the development of drugs, pharmaceutical agents, and even in the use of chemicals. [0102] The following Examples are provided to illustrate but not limit the present invention. It will be apparent to one of skill in the art that modifications can be made while keeping in the spirit and scope ofthe present invention.

[0103] EXAMPLE 1 : PRIMER DESIGN

[0104] Primers were designed by using the web page: http://www- genome.wi.mit.edu (scroll down to find the link: WWW Primer Picking

{Primer3}). Thus, Primer3 software was used to pick the primers based on inputted parameters such as melting temperature and length. The following factors were used for the design ofthe primers:

[0105] 1. Global parameters, product size range, which depends on the size of the equivalent rat target on the CT array. For example: if it is known that the rat target is 500 bases long, then 480-520 should be filled in as the range for a possible size for the human target.

[0106] 2. Per sequence inputs, region included: The appropriate region for the human target sequence is known from the homology search between the human and the rat genes (full length). Fill in the base number where the computer should start looking for primers followed by the approximate length: e.g., 2697, 550

[0107] 3. Global parameters, Tm minimum: 65 °C

[0108] 4. Global parameters, Tm maximum: 72°C

[0109] 5. Global parameters, Tm optimum: 69°C

[0110] 6. Global parameters, Minimum primer length: 23

[0111] 7. Global parameters, Maximum primer length: 27

[0112] 8. Global parameters, Optimum primer length: 25

Primer concentrations and storage

[0113] Primers were ordered from Genset (Paris, France). Oligonucleotides were supplied as a lyophilized powder. The powder was dissolved in nanopure water to obtain a stock solution, and then the stock solution was diluted to a working concentration of lOpmol/μl (lOμM).

[0114] The following procedures were followed:

[0115] 1. Briefly spin tube containing the lyophilized powder to make sure all powder is at the bottom ofthe tube.

[0116] 2. Completely dissolve the oligonucleotide in the appropriate amount of nanopure water to obtain a stock solution of lnmol/μl (lmM). [0117] 3. Dilute 1 μl of this stock solution with 99μl of nanopure water to obtain a working solution of lOpmol/μl (lOμM).

[0118] 4. Label the tube with working solution with the oligo number and the concentration (lOμM) and store at -20°C.

[0119] 5. Dry down the rest ofthe stock solution in the Speedvac. Store the dried powder at -20°C. For example: The original amount of oligo was dissolved in 10.5μl of water to obtain the lmM stock solution. This stock was then diluted

1 μl in lOOμl to make the working solution and then dried down the rest ofthe stock. Next time the powder would be dissolved in 9.5 μl of water to prepare the lmM stock solution.

EXAMPLE 2: POLYMERASE CHAIN REACTION (PCR)

[0120] AdvanTaq PCR kit from Clontech was used. This kit contains the 10 X

PCR buffer, 50 X dNTPs, PCR grade water and the 50 X Taq polymerase. The kit was stored at -20°C as recommended.

[0121] The following procedure was followed:

[0122] 1. Place all the components on ice and allow them to thaw. Water and

10 X buffer can be thawed at room temperature.

[0123] 2. Combine the following reagents in a 0.5 ml PCR tube on ice:

PCR grade water 38μl

10 X PCR buffer 5μl

DNA template (Clontech QUICK-Clone™ cDNA) 1 μl

Forward Primer (lOμM) 2μl

Reverse Primer (lOμM) 2μl

50 X dNTP Mix (1 OmM each dNTP) 1 μl

50 X Taq DNA polymerase lμl

TOTAL 50μl

[0124] 3. Mix by pipeting up and down several times. If multiple PCR reaction will be performed simultaneously, a PCR master mix can be prepared.

[0125] 4. To separate labelled 0.5ml PCR tubes, add the appropriate forward and reverse primers.

Forward Primer (lOμM) 2μl Reverse Primer (lOμM) 2μl

[0126] 5. Per 10 reactions, combine the following reagents on ice:

PCR grade water 399μl

10 X PCR buffer 52.2μl

DNA template lOμl

50 X dNTP Mix 10.5μl

50 X Taq DNA polymerase lO.Sμl

[0127] 6. Mix by pipeting up and down.

[0128] 7. To each tube, add 46 μl ofthe master mix. Mix by pipeting.

[0129] 8. Program the thermocycler to run the following program:

95°C for 1 minute -» 35 cycles 95°C for 45 seconds -» 63°C for 45 seconds - 68°C for 1 minute - 68°C for 10 minutes. [0130] 9. Place the tubes in the thermocycler and start the program. [0131] 10. When the PCR reaction is finished, an aliquot is run on an agarose gel to verify if the desired cDNA fragment was made. 0.5M EDTA pH 8.0 (500ml) is made by dissolving 93 g of Na₂EDTA.2H₂O in 400ml of nanopure water, and adjusting pH to 8.0 with NaOH. EDTA will not dissolve completely until pH is adjusted. Add water to 50 ml. Autoclave. 10 X TBE (1 liter) is made by using 108g Tris base, 55g boric acid, 40ml of 0.5M EDTA pH 8.0 (or 7.4g Na₂EDTA.2H₂O). 10 mg/ml ethidium bromide is made by dissolving one tablet of ethidium bromide (Merck) in 10 ml of nanopure water.

[0132] 11. While the PCR program is running, pour a 1.2% agarose gel. In an Erlenmeyer or glass bottle, add the following material: 1.8 g agarose, 15 ml 10 X TBE, and 135 ml water. Dissolve the agarose by heating the mixture in the microwave oven. Let cool down to approximately 65°C. Add 1.5 - 3 μl of ethidium bromide (10 mg/ml). Pour the gel in the tray, insert combs, and let solidify at room temperature. When the PCR program is finished, take the tubes out ofthe machine and put on ice. From each tube, transfer 5μl to a new-labelled tube and add 1 μl 6 X Loading dye. Load the samples on tlie agarose gel. Include at least one lane with DNA molecular weight marker (Eurogentec Smart Ladder; use 5μl per lane). Immediately freeze the rest ofthe PCR reaction at -20°C. Run the gel in 1 X TBE at 100V until the blue dye has migrated a few centimeters in the gel. Analyze the gel on UV light and take a Polaroid picture. If you see multiple bands in a lane, then the PCR product needs to be gel-purified. If only one band is visible, the PCR can be used as such for cloning. If the PCR product needs to be purified, run another agarose gel and load 30 μl ofthe PCR reaction (+ 6 μl 6 X loading buffer). Run the gel. Visualize band on UV light. [0133] 12. Using a clean and sharp scalpel cut ofthe band corresponding to the desired length. Put the piece of agarose gel in an Eppendorf tube and either proceed immediately to the clean-up, or store the gel at -20°C.

DNA cleanup from agarose gel

[0134] This procedure is only necessary when the PCR reaction contains multiple products. If only one band (ofthe correct length) is visible on agarose gel, this procedure can be omitted.

[0135] 1. Use QIAquick 8 PCR Purification kit and QIAGEN buffer QG with the QIAvac S6 manifold. Add ethanol to buffer PE before use. Label the bottle after ethanol is added. [0136] 2. Excise the desired DNA fragment from the agarose gel using a clean scalpel.

[0137] 3. Weigh the gel slice.

[0138] 4. Add three volumes of buffer QG to one volume of gel (lOOmg =

100ml)

[0139] 5. Incubate at 50°C for 10 minutes to dissolve the agarose. Vortex every 2-3 minutes to help dissolve the gel.

[0140] 6. In the meantime, assemble the QIAvac S6 manifold. Open the S6 lid and place the required amount of 8-well strips in the slots. Seal any unused slots with blanks and close the QIAvac S6 lid. Check the vacuum: it should be between -200 to -600mbar.

[0141] 7. After the gel is dissolved, make sure that the color ofthe mixture is yellow. If the color is orange or violet, add lOμl of 3M sodium acetate pH 5.0 to adjust the pH. The color ofthe mixture should now turn yellow.

[0142] 8. Add one gel volume of isopropanol to the sample and mix.

[0143] 9. Apply the samples to the wells ofthe QIAquick strips. Switch on the vacuum source. After all the liquid has been pulled through, switch off the vacuum.

[0144] 10. Add 0.5ml of buffer QG. Apply vacuum. After all liquid has been pulled through, switch off vacuum.

[0145] 11. Add 1ml of buffer PE to each column. Pull through by applying vacuum.

[0146] 12. Repeat previous step. After buffer PE has been pulled through, apply maximum vacuum for an additional 5 minutes to dry the membrane. Switch off the vacuum source and ventilate the QIAvac S6 slowly. Remove the top plate ofthe QIAvac S6 (containing the 8-well strips) and vigorously rap the plate on a stack of absorbent papers, until no drops come out.

[0147] 13. Replace the waste tray with a rack of collection tubes in the

QIAvac S6. Place the top plate (+ columns) back on the base. [0148] 14. Add 60μl of buffer EB or nanopure water to the center ofthe membrane in each column. Let stand for 1 minute. Switch on the vacuum for 5 minutes to elute the DNA. [0149] 15. Ventilate the QIAvac slowly.

EXAMPLE 3: TOPO TA CLONING OF CDNA FRAGMENTS

[0150] The PCR-amplified cDNA fragments is cloned in the Stratagene pCR^®II-TOPO^® vector. If the PCR reaction contained only one cDNA fragment (ofthe desired length), it can be cloned without purification. If not, the desired band is purified prior to cloning using the QIAquick PCR purification kit. It is important to use a thermostable polymerase during PCR with a non-template- dependent terminal transferase activity that adds a single deoxyadenosine (A) to the PCR product. The vector supplied in the kit is linear and has a single T overhang. This allows the PCR product to ligate efficiently within the vector. [0151] 1. The ligation reaction is accomplished at room temperature as follows:

PCR product (total reaction or purified) 0.5 to 4.0μl Salt solution lμl

Sterile water add to total Volume of 5μl

TOPO vector lμl

Final volume 6μl

[0152] All the components are stored at -20°C, except the salt solution and the water, which can be stored at 4°C; thaw the components on ice before use. [0153] 2. Mix the reaction gently and incubate at room temperature for 5 minutes. Immediately proceed to the transformations. Alternatively, the reactions can be put on ice until all ligations are completed. The reactions can also be stored at -20°C overnight.

EXAMPLE 4: TRANSFORMATIONS

[0154] Chemical transformation protocol was used. An alternative method of transformation which can be used is electroporation. Chemical transformation requires One Shot Chemically competent E.coli strain TOP 10 bacteria. The following solution were made and used for the transformation:

AmpiciUin stock solution dOOmg/mD

• Dissolve 1 g of ampiciUin (sodium salt) in 10ml of nanopure water

• Filter sterilize through a 0.2μm filter

• Store at -20°C

X-gal stock solution (4% w/v)

• Dissolve 500mg of X-gal in 12.5ml of DMF (dimethylformamide)

• Store at -20°C, protected from light (in a brown bottle)

IPTG stock solution (TOOn M

• Dissolve 238mg of IPTG in 10 ml of nanopure water

• Filter sterilize (0.2μm filter) and store in 1ml aliquots at -20°C

LB/amp/X-gal plates (1L = +/- 30 plates^{^})

• Dissolve 40 grams of Luriah/agar medium in 950 ml of water. Add water to IL.

• Autoclave on liquid cycle for 20 minutes at 15 psi.

• Allow the solution to cool down to 55°C.

• Add ampiciUin to a final concentration of 1 OOμg/ml (1 ml of stock per liter).

• Add 2 ml of X-gal (4%) to 1 L of medium.

• Pour into 10cm sterile petri dishes (approx. 3ml per plate).

• Allow cooling down.

• Invert and store at 4°C.

[0155] 1. Be sure to have enough LB/amp/X-gal plates at 37°C.

[0156] 2. Equilibrate a water bath to 42°C.

[0157] 3. Warm the SOC medium to room temperature.

[0158] 4. Tha on ice one vial of chemically competent One Shot cells per transformation.

[0159] 5. Add 2μl ofthe TOPO cloning reaction to a vial of competent cells and mix gently. Do not mix by vortexing or pipeting up and down.

[0160] 6. Incubate on ice for 20 minutes.

[0161] 7. Heat shock the cells at 42°C for 30 seconds without shaking.

[0162] 8. Immediately put the tubes on ice and incubate for an additional 5 minutes. [0163] 9. Add 250μl of room temperature SOC medium and shake the tube horizontally at 37°C (200rpm) for 1 hour.

[0164] 10. Spread 200μl ofthe transformation reaction on selective plates. Incubate overnight at 37°C

EXAMPLE 5: ANALYSIS OF POSITIVE CLONES

[0165] Using PCR, a minimum of three colonies from each transformation are analyzed for the correct cDNA insert. Either the Ml 3 Forward and Reverse primer set supplied with the cloning kit is used, or the T7 and Sp6 primer set made in-house. These primers anneal to the vector and amplify any insert in the vector. The resulting PCR products are analyzed on an agarose gel and the length ofthe insert is compared to the length ofthe cloned fragment. Prior to PCR analysis, the colonies will be streaked on a separate selective LB/amp plate. The following steps are used:

[0166] 1. Pick separate white or light blue colonies from the transformation plates (minimum 3 colonies per plate) with a sterile toothpick.

[0167] 2. Resuspend the clones individually in 20μl of sterile nanopure water.

[0168] 3. Streak the colonies on a fresh, correctly labelled LB/agar plate containing ampiciUin.

[0169] 4. Incubate the plate overnight at 37°C

[0170] 5. Incubate the tubes prepared in step 2 at 96°C for 10 minutes to lyse the cells and inactivate nucleases. Spin briefly.

[0171] 6. Prepare the PCR mix on ice. For each reaction, add the following components (multiply by the amount of reactions to be performed; also be sure to prepare some in excess). The following materials are used:

PCR water 36.5μl 400μl (for 10 reactions)

10 X PCR buffer 5μl 55μl (for 10 reactions)

Forward primer (Ml 3F) (10 μM) 1 μl 11 μl (for 10 reactions)

Reverse Primer (Ml 3R) (10 μM) lμl 1 lμl (for 10 reactions) dNTP mix (10 mM each) 1 μl 11 μl (for 10 reactions) Taq DNA polymerase 0.5μl 5μl (for 10 reactions)

[0172] 7. Mix by pipeting up and down.

[0173] 8. To a set of labelled PCR tubes, add 5μl ofthe resuspended clones.

[0174] 9. To each tube, add 45μl ofthe PCR mix.

[0175] 10. Place the tubes in a thermocycler, program the following parameters:

96°C for 3 minutes -> 25 cycles of a). 96°C for 1 minute b). 55°C for 1 minute c). 72°C for 1 minute ^■» 72°C for 10 minutes -» hold at 4°C. [0176] When the program is finished, add 4μl of 6 X loading buffer to 20μl of each sample and analyze the PCR product on a 1.2% agarose gel. The rest ofthe reaction is stored at -20°C.

EXAMPLE 6: IDENTIFYING AND ISOLATING GENES INVOLVED IN TOXICOLOGICAL RESPONSES

[0177] Human liver cells exposed to a toxic dose of aflatoxin (1 mg/ml) or liver tissue from a human exposed to aflatoxin are used to determine the differentially expressed genes in a human exposed to a liver toxicant (aflatoxin). Exposure to a toxicant can occur intentionally (e.g., clinical treatment) or unintentionally (e.g., occupational exposure or accidental exposure). RNA is isolated from both liver samples using an RNA isolation kit from Qiagen (RNeasy Midi kit) followed by use of a MessageClean^® kit from Genhunter^®. The protocols from the MessageClean^® kit are modified to generate more optimal conditions for removing DNA contamination. Then, these ingredients are added: 50 μl total RNA, 5.7 μl lOx reaction buffer, 1.0 μl DNase I (10 units/μl) for a total volume of 56.7 μl. The ingredients are mixed well and incubated for 30 minutes at 37° Celsius. Then 40 μl phenol/chloroform mixture (1:1 volume) is added and the mixture is vortexed for 30 seconds and allowed to sit on ice for 10 minutes. Then the tube containing the mixture is spun in an Eppendorf centrifuge at 4 degrees for 5 minutes at maximum speed. The upper phase is collected, transferred to a new tube and 5 μl of 3M NaOAc and 200 μl 95% ethanol is added to the upper phase. The mixture is allowed to sit for at least one hour at -80° C and then spun for about 10 minutes at 4° C. The supernatant is removed and the RNA dried for a few minutes. Subsequently, the RNA is suspended in 11 μl DEPC H₂0. 1 μl is used to measure A₂₆o_/280 in 50 μl H₂0. The RNA is stored as 1- 2 μg aliquots at -80°C. Immediately prior to differential display, the appropriate amount of RNA is diluted to 0.1 μg/μl with DEPC H₂0 . It is important to avoid using the diluted RNA after freeze-thaw cycle.

[0178] RNAimage^® kits are used and protocols from the RNAimage^® kits are altered to optimize more successful mRNA differential display. The following sections describe the methods by which this is accomplished:

Reverse transcription

[0179] In a tube, the following ingredients are added: 9.4 μl dH₂0, 4.0 μl 5x RT buffer, 1.6 μl dNTP (250 μM), 2.0 μl of 0.1 μg/μl freshly diluted total RNA that is DNase-free, 2.0 μl H-TπM (2 μM) for a total volume of 19 μl. The ingredients are mixed well and incubated at 65°C for 5 minutes, 37°C for 60 minutes, 75°C for 5 minutes, and held at 4°C. After the tubes had been at 37°C for 10 minutes, and 1 μl of Superscript II reverse transcriptase (Life Technologies Inc.) is added to each reaction, and quickly mixed by finger tapping the tubes before the incubation continued. At the end ofthe reverse transcription, the tubes are spun briefly to collect condensation. The tubes are set on ice for PCR or stored at -20°C for later use.

PCR

[0180] The following ingredients are used for a PCR reaction: 10 μl dH₂0, 2 μl 10X PCR buffer, 1.6 μl dNTP (25 μM), 2 μl of 2 μM H-AP primer, 2 μl of 2 μM H-TπM, 2 μl RT-mix described above (must contain the same H-TπM used for PCR), 0.2 μl α-³³P dATP (2000 Ci/mmole), 0.2 μl Taq DNA polymerase from PE Biosystems for a total volume of 20 μl. The tube containing all these ingredients are mixed well by pipeting up and down and placed in a thermocycler at 95°C for 5 minutes and then amplified for 40 cycles under the conditions of 94°C for 30 seconds, 40°C for 2 minutes, 72°C for 30 seconds and finally held at 4°C until the samples are removed from the thermocycler.

Gel electrophoresis

[0181] A 6% denaturing polyacrylamide gel in TBE is prepared and allowed to polymerize for at least 2 hours before using. Then the gel is run for about 30 minutes before any samples are loaded. It is important for all the sample wells in the gel to be flushed and cleared of all urea prior to loading any samples in the wells. About 3.5 μl of each sample is mixed with 2 μl of loading dye and incubated at 80°C for 2 minutes immediately before loading onto the 6% gel. In this example, the loading dye is xylene and after the gel is loaded with the samples obtained from the rounds of PCR, the gel is run at 60 watts of constant power until the xylene dye is about 6 inches from the bottom ofthe gel. Once the power is turned off, the gel is blotted onto a large sheet of exposed autoradiograph film. The gel is covered with plastic wrap and under dark conditions, the gel is placed in a large autoradiograph cassette with a new sheet of unexposed film, marked for orientation, and the film is allowed to be exposed to the gel at -80°C The exposure period can be anywhere from overnight to 72 hours. Once the film has been developed, bands of interest are identified by alignment with the developed film and subsequently isolated by cutting the band of interest out ofthe polyacrylamide gel with a clean scalpel blade. The isolated band is placed in 100 μl of water and boiled at 95% for 5 minutes.

PCR to amplify gel band

[0182] PCR is set up to amplify the gel band. The re-amplification should be done using the same primer set and PCR conditions except the dNTP concentrations should be at 20 μM. The following ingredients are combined for the PCR reaction: 20.4 μl H₂0, 4 μl 10X PCR buffer, 3.2 μl of 250 μM dNTPs , 4 μl of 2 μM H-AP primers, 4 μl of 2 μM H-T_πM, 4 μl template (out ofthe 100 μl containing gel band), and 0.5 μl Taq polymerase for a total volume of 40 μl. These ingredients are heated to 95°C for 5 minutes and then cycled for 40 cycles under the conditions of 94°C for 30 seconds, 40°C for 2 minutes, 72°C for 30 seconds followed by a final extension at 72°C for 5 minutes and finally held at 4°C until the samples are removed from the thermocycler. About 4 μl ofthe PCR reaction is removed and run on a 1% agarose gel to ascertain the success ofthe PCR reaction.

Cloning amplified fragments

[0183] To clone the amplified fragments, products from different sources (e.g., GenHunter® or InVitrogen) may be used to achieve the desired cloned product. In this example, InVitrogen' s TOPO TA Cloning Kit® is used and the following material is combined in a reaction tube: 2 μl of freshly run PCR product, 2 μl of sterile H₂0, 1 μl of PCR-TOPO vector for a final volume of 5 μl. The combined ingredients are mixed gently and incubated for 5 minutes at room temperature. Then 1 μl of 6x TOPO Cloning Stop Solution is added and all combined ingredients are mixed for about 10 seconds at room temperature and then set on ice. One Shot™ cells are thawed on ice. 2 μl ofthe TOPO Cloning reaction is added to the One Shot™ cells, mixed, and incubated on ice for 30 minutes. The cells are heat shocked at 42°C for 30 seconds without shaking and incubated on ice for 2 minutes. Then 250 μl of room temperature SOC is added to the heat shocked cells and mixed. The cells are then placed at 37°C for 30 minutes. About 50-100 μl ofthe cells are spread on 2 XYT plates containing 100 μg/ml ampiciUin and X-gal. The plates are incubated overnight at 37°C and the next morning, 3 white colonies are selected for analysis.

Screening colonies for correct recombinant plasmids

[0184] PCR is used to ascertain whether the white colonies selected contained the correct recombinant plasmid. The following ingredients are combined for the PCR reaction: 21 μl H₂0, 2.5 μl 10X PCR buffer, 0.12 μl of lOmM dNTPs, 1 μl of 25 ng/μl T7 primer, 1 μl gene specific left or right primer at 25 ng/μl, template (a toothpick is used to transfer colony from transformation plate to tube by swishing the toothpick around in the reaction mix), and 0.5 μl Taq polymerase for a total volume of 25 μl. The reaction mix is run at 95°C for 5 minutes and then cycled 35 times under the conditions of 95° C for 30 seconds, 45°C for 30 seconds, 72° C for 30 seconds, and followed by 72° C for 5 minutes and finally 4°C until samples are removed from the thermocycler. About 4 μl ofthe PCR product is removed and run on a 1% agarose gel to ascertain the success ofthe PCR reaction. Bacterial colonies corresponding to the colonies which yielded positive PCR results are grown overnight in LB media containing 100 μg/μl ampiciUin at 37° C with constant shaking. Plasmid DNA are isolated from the overnight cultures and sequenced using a T7 primer. Sequences are then compared to sequences in the GenBank database to confirm that the correct gene fragment is cloned. Gene fragments are then amplified by PCR from the plasmid DNA. The unincorporated primers and dNTPs are removed and the resulting gene fragments are arrayed on glass slides for the purposes of measuring differential gene expression using the Phase- 1 Molecular Toxicology Microarray products.

EXAMPLE 7: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT GENES FROM HUMAN DATABASES

[0185] One method that was used to identify and isolate toxicologically relevant genes for inclusion in a human array was to search a public database (e.g., GenBank) for toxicologically relevant human genes. Once these genes were identified, primers, for example, those listed in Table 2 were designed and used in an amplification process with a cDNA library made from human cells. As disclosed herein, a cDNA library can be made from a variety of sources including but not limited to liver, lymphocytes, spleen, lung, kidney, brain, thymus, heart, tissue culture cells, primary cells, lymph nodes, or obtained from a commercial source (e.g., Clontech QUICK-Clone™ Cat. No. 7109-1). The amplified product was cloned into an expression vector and sequenced to confirm that the sequence matched or was substantially similar to the gene sequence information obtained from GenBank. Confirmed amplified gene products were then incorporated into a human array using the methods disclosed herein to immobilize the gene product, or target sequence, to a glass slide. EXAMPLE 8: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT GENES FROM RAT HOMOLOGUES

[0186] One method that is used to identify and isolate toxicologically relevant genes for inclusion in a human array is to make primers to toxicologically relevant rat genes, for example, as disclosed in pending U.S. applications 60/264,933 and 60/308,161. Once toxicologically relevant rat (or other non-human species) genes are identified, human homologues are identified by searching human sequence database (e.g., GenBank) for human sequence homologous to the non-human gene sequences with sequence search tools such as the Basic Local Alignment and Search Tool (Alschul et. al. 1997). Primers are obtained and used in an amplification process with cDNA library made from human cells. As disclosed herein, cDNA library can be made from a variety of sources (e.g., liver, lymphocytes, etc.). Confirmed amplified gene products are then incoφorated into a human array using the methods disclosed herein to immobilize the gene product, or target sequence, to a glass slide. Primers which were used to obtain human toxicologically relevant genes which are homologues of toxicologically relevant rat genes are disclosed in Table 2.

EXAMPLE 9: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT HUMAN GENES USING RAT HOMOLOGUES

[0187] Sequences of human homologues of a toxicologically relevant rat gene sequence were obtained by using the sequence of toxicologically relevant rat gene sequences in a sequence search (e.g., a BLAST search) to find human sequences which have high homology to the rat gene. Primers to the human homologue were synthesized and then used to amplify a sequence ofthe human homologue from a human cDNA library as detailed in previous examples. Table 2 lists the primers which were used to isolate the human homologues. Table 5 lists the sequences obtained from cloned human homologues of rat genes prepared by this approach. EXAMPLE 10: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT GENES USING DENOVO PRIMERS

[0188] Toxicologically relevant genes are identified using a public database (e.g., GenBank) and sequences corresponding within these genes are synthesized de novo and used in amplification reactions. The amplified product was cloned into a cloning vector and sequenced to confirm that the sequence matched or was substantially similar to the gene sequence information obtained from GenBank. Confirmed amplified gene products were then incorporated into a human array using the methods disclosed herein to immobilize the gene product, or target sequence, to a glass slide.

EXAMPLE 11: ATTACHINGTOXICOLOGICALLYRELEVANT GENES

TO GLASS SLIDES

[0189] The genes to be attached to the glass slides can be amplified as provided herein. An important modification to the amplification process was the inclusion of amine primers, which can be obtained from any commercial source, e.g., Synthegen, such that a reactive amine group, a derivative thereof, or another reactive group was included in the amplified product. The amplified product was purified by any number of methods disclosed herein and immobilized or "spotted" onto a solid substrate, such as a glass slide, which can react with the amine group on the amplified product and form a covalent linkage.

MD Array Spotter Operation

[0190] The terminology and equipment used in this example comprised the following:

[0191] Spotter: MD Generation II Array Spotter main instrument

[0192] Spotting Chamber: Area of spotter enclosed in glass which houses the pins, plates, trays and most spotter machinery.

[0193] Controller: Dedicated Dell Computer and Monitor to right of Spotter

Unit [0194] Pins: (6) fine tubes in the Spotter Unit which pick-up and spot the Target

[0195] Slides: Std. size glass microscope slides with a special coating on one side

[0196] Plates: Plastic 96 well plates which hold the Target solution to be spotted

[0197] Target: A solution of PCR product which the spotter deposits on the slides.

[0198] N2 Tank: 5 ft. high steel gas tank labeled "Nitrogen, Compressed"

[0199] N2 : The N2 gas from the N2 tank

[0200] Air Conditioner: Kenmore air conditioner installed in window of spotting chamber

[0201] Humidifier 1 : Essick 2000 Evaporative Cooler against the window [0202] Humidifier 2: Bemis Airflow with white flexible duck into the Spotter Unit [0203] Humidifier 3: Bemis Airflow against the wall

[0204] Humidifier 4: Kenmore QuietComfort 7 [0205] Vacuum Pump: Gast Laboratory Oilless Piston Vacuum Pump [0206] Dampbox: The plastic sealable container containing an NaCl / water slurry

[0207] Materials used for reagent solutions were: Nanopure water, 0.2 M KCI

(1/10 dilution of Stock 2M KCI in water), and 95% EtOH

Reagent. The temperature control was adjusted to 60°C. The spotter chambers were adjusted to be greater than 39 % relative humidity and less than

65° C. The spotting pins were pre-washed for 20 cycles.

Slide Preparation/Loading:

[0208] When the pre- wash was completed, the slides were first each blown with N2 gas for about 2 seconds per side. The slides were inserted into the Spotter following Array Spotter Run Values. The slides were aligned using a clean narrow rod orienting it on the center right edge ofthe slide and gently pushed to the left until the slide was aligned vertically against the metal pins. After slides were loaded and straightened, a visual check was done to make sure no more debris had fallen. The humidity was confirmed to be greater than 39% relative humidity. The MD spotter recognizes 16 plates as a maximum for a run and will pause automatically after 8 plates. The MD spotter also advances sequentially to plates in an invariable order and is not programmable to accommodate unique plate sourcing scheme. Therefore, it was important to manually rotate (or shuffle) plates to accomplish the spotting for the human arrays.

Blocking (Slide Preparation post-spotting)

[0209] This blocking procedure is important because it reduces the nonspecific background signals. The amounts provided in this protocol are for 19 slides, however, a skilled artisan may make modifications accordingly. More staining dishes and slide racks will be required if more than 19 slides are to be blocked. A clean glass container was obtained and filled with Nanopure H20. The container was placed on a hot plate and heated to a high temperature. A blocking solution was made by adding 2.5 ml of 20% SDS to 500mL blocking solution bottle. The blocking solution was warmed in microwave for 2.5 minutes and checked to determine if the temperature had reached 50°C If the temperature of the solution was not at yet 50°C, then the solution was warmed in the microwave at 10 second intervals until it reached the desired temperature. One staining dish was placed on an orbital shaker with 4x SSC solution and turned to an agitation speed of 75 rpm. Slides were placed in metal racks and placed in boiling water for several minutes (e.g., 2 minutes). The slides were taken out of boiling water and allowed to cool briefly. The slides were then transferred to staining container containing 4x SSC solution on orbital shaker for several minutes (e.g., 2 minutes), rinsed with nanopure water in a staining container, and then briefly placed in blocking solution for about 15 minutes. After 15 minutes, the slides were taken out ofthe blocking solution and rinsed three times by dipping into three separate containers with nanopure water each time. The tops ofthe slides were dabbed lightly with a tissue and the slides were placed in a centrifuge for about 5 minutes at a speed of 1000 rpm.

EXAMPLE 12: MICROARRAY RT REACTION

[0210] Fluorescence-labeled first strand cDNA probe was made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra. This probe is hybridized to microarray slides spotted with DNA specific for toxicologically relevant genes. The materials needed to practice this example are: total or messenger RNA, primer, Superscript II buffer, dithiothreitol (DTT), nucleotide mix, Cy3 or Cy5 dye, Superscript II (RT), ammonium acetate, 70% EtOH, PCR machine, and ice.

[0211] The volume of each sample that would contain 20μg of total RNA (or 2μg of mRNA) was calculated. The amount of DEPC water needed to bring the total volume of each RNA sample to 14 μl was also calculated. If RNA is too dilute, the samples are concentrated to a volume of less than 14 μl in a Speedvac without heat. The Speedvac must be capable of generating a vacuum of 0 Milli- Torr so that samples can freeze dry under these conditions. Sufficient volume of DEPC water was added to bring the total volume of each RNA sample to 14 μl. Each PCR tube was labeled with the name ofthe sample or control reaction. The appropriate volume of DEPC water and 8 μl of anchored oligo dT mix (stored at - 20°C) was added to each tube.

[0212] Then the appropriate volume of each RNA sample was added to the labeled PCR tube. The samples were mixed by pipeting. The tubes were kept on ice until all samples are ready for the next step. It is preferable for the tubes to kept on ice until the next step is ready to proceed. The samples were incubated in a PCR machine for 10 minutes at 70°C followed by 4°C incubation period until the sample tubes were ready to be retrieved. The sample tubes were left at 4°C for at least 2 minutes. [0213] The Cy dyes are light sensitive, so any solutions or samples containing Cy-dyes should be kept out of light as much as possible (e.g., cover with foil) after this point in the process. Sufficient amounts of Cy3 and Cy5 reverse transcription mix were prepared for one to two more reactions than would actually be run by scaling up the following recipes:

For labeling with Cy3

[0214] 8 ul 5x First Strand Buffer for Superscript II

[0215] 4 ul 0.1 M DTT

[0216] 2 ul Nucleotide Mix

[0217] 2 ul of 1 :8 dilution of Cy3 (i.e., 0.125mM Cy3dCTP).

[0218] 2 ul Superscript II

For labeling with Cy5

[0219] 8 ul 5x First Strand Buffer for Superscript II

[0220] 4 ul 0.1 M DTT

[0221] 2 ul Nucleotide Mix

[0222] 2 ul of 1:10 dilution of Cy5 (i.e., O.lmM Cy5dCTP).

[0223] 2 ul Superscript II

[0224] About 18 μl ofthe pink Cy3 mix was added to each treated sample and 18 μl ofthe blue Cy5 mix was added to each control sample. Each sample was mixed by pipeting. The samples were placed in a PCR machine for 2 hours at 45°C followed by 4°C until the sample tubes were ready to be retrieved. The samples were transferred to Eppendorf tubes containing 600 μl of ethanol precipitation mixture. Some ofthe EtOH precipitation mixture was used to rinse the PCR tubes. The tubes were inverted to mix. Samples were placed in -80°C freezer for at least 20-30 minutes. If desired, samples may be left at -20°C overnight or over the weekend.

[0225] The samples were centrifuged for 15 minutes at 20800 x g (14000 rpm in Eppendorf model 5417C) and carefully the supernatant was decanted. A visible pellet was seen (pink/red for Cy3, blue for Cy5). It is a preferable to centrifuge the tubes at a fixed position so the pellet will be at a known area in the tube. In some rare instances, the probe is seen spread on one side ofthe tube instead of a tight pellet. If the pellet is white or nonexistent, the reaction has not occurred to maximal efficiency.

[0226] Ice cold 70% EtOH (about 1 ml per tube) was used to wash the tubes and the tubes were subsequently inverted to clean tube and pellet. The tubes were centrifuged for 10 minutes at 20800 x g (14000 rpm in Eppendorf model 5417C), then the supernatant was carefully decanted. The tubes were flash spun and any remaining EtOH was removed with a pipet. The tubes were air dried for about 5 to 10 minutes, protected from light. The length of drying time will depend on the natural humidity ofthe environment. For example, an environment in Santa Fe would require about 2 to 5 minutes of drying time. It is preferable that the pellet are not overdried.

[0227] When the pellets were dried, they are resuspended in 80 ul nanopure water. The cDNA/mRNA hybrid was denatured by heating for 5 minutes at 95°C in a heat block and flash spun.

EXAMPLE 13: PURIFICATION OF CY -DYE LABELED CDNA

[0228] To purify fluorescence-labeled first strand cDNA probes, the following materials were used: Millipore MAHV N45 96 well plate, v-bottom 96 well plate (Costar), Wizard DNA binding Resin, wide orifice pipette tips for 200 to 300 μl volumes, isopropanol, nanopure water. It is highly preferable to keep the plates aligned at all times during centrifugation. Misaligned plates lead to sample cross contamination and/or sample loss. It is also important that plate carriers are seated properly in the centrifuge rotor.

[0229] The lid of a "Millipore MAHV N45" 96 well plate was labeled with the appropriate sample numbers. A blue gasket and waste plate (v-bottom 96 well) was attached. Wizard DNA Binding Resin (Promega cat#Al 151) was shaken immediately prior to use for thorough resuspension. About 160 μl of Wizard DNA Binding Resin was added to each well ofthe filter plate that was used. If this was done with a multi-channel pipette, wide orifice pipette tips would have been used to prevent clogging. It is highly preferable not to touch or puncture the membrane ofthe filter plate with a pipette tip. Probes were added to the appropriate wells (80 μl cDNA samples) containing the Binding Resin. The reaction is mixed by pipeting up and down ~10 times. It is preferable to use regular, unfiltered pipette tips for this step. The plates were centrifuged at 2500 rpm for 5 minutes (Beckman GS-6 or equivalent) and then the filtrate was decanted. About 200 μl of 80% isopropanol was added, the plates were spun for 5 minutes at 2500 rpm, and the filtrate was discarded. Then the 80% isopropanol wash and spin step was repeated. The filter plate was placed on a clean collection plate (v-bottom 96 well) and 80 μl of Nanopure water, pH 8.0-8.5 was added. The pH was adjusted with NaOH. The filter plate was secured to the collection plate with tape to ensure that the plate did not slide during the final spin. The plate sat for 5 minutes and was centrifuged for 7 minutes at 2500 rpm. If there are replicates of samples they should be pooled.

EXAMPLE 14: FLUORESCENCE READINGS OF CDNA PROBE AND HYBRIDIZATION ON THE MICROARRAY

[0230] To semi-quantitatively assess the incorporation of fluorescence into cDNA probes and to concentrate probes prior to hybridization, the following material was used: 384 well, 100 μl assay plate (Falcon Microtest cat#35-3980) and Wallac Victor 1420 Multilabel counter (or equivalent). [0231] It is preferable that a consistent amount of cDNA is pipeted into the 384-well plate wells because readings will vary with volume. Confrols or identical samples should be pooled at this step, if required. The probes were transferred from the Millipore 96 well plate to every other well of a 384 well assay plate (Falcon Microtest). This was done using a multi-channel pipette. For replicate samples that have been pooled, 60 μl aliquots were transferred into wells ofthe assay plate.

[0232] The Cy-3 and Cy-5 fluorescence was analyzed using the Wallac 1420 workstation programmed for reading Cy3-Cy-5 in the 384-well format and the data was saved to disk. The typical range for Cy-3 (20μg) is 250-700,000 fluorescence units. The typical range for Cy-5 (20μg) is 100-250,000 fluorescence units. Settings for the Wallac 1420 fluorescence analyzer were as follows:

Cy3

[0233] CW lamp energy = 30445

[0234] Lamp filter = P550 slot B3

[0235] Emission filter = D572 dysprosium slot A4

[0236] Emission aperture = normal

[0237] Count time = 0.1 s

Cy5

[0238] CW lamp energy = 30445

[0239] Lamp filter = D642 samarium slot B7

[0240] Emission filter = D670 slot A8

[0241] Emission aperture = normal

[0242] Count time = 0.1 s

Dry-down Process

[0243] Concentration ofthe cDNA probes is highly preferable so that they can be resuspended in hybridization buffer at the appropriate volume. The volume of the control cDNA (Cy-5) was measured and divide by the number of samples to determine the appropriate amount to add to each test cDNA (Cy-3). Eppendorf tubes were labeled for each test sample and the appropriate amount of control cDNA was allocated into each tube. The test samples (Cy-3) were added to the appropriate tubes. These tubes were placed in a speed- vac to dry down, with foil covering any windows on the speed vac. At this point, heat (45°C) may be used to expedite the drying process. Time will vary depending on the machinery. The drying process takes about one hour for 150 μl samples dried in the Savant. Samples may be saved in dried form at -20°C for up to 14 days. Microarray Hybridization

[0244] To hybridize labeled cDNA probes to single stranded, covalently bound DNA target genes on glass slide microarrays, the following material were used: formamide, SSC, SDS, 2 μm syringe filter, salmon sperm DNA, hybridization chambers, incubator, coverslips, parafilm, heat blocks. It is preferable that the array is completely covered to ensure proper hybridization. [0245] About 30 μl of hybridization buffer was prepared per sample. Slightly more than is what is needed should be made since about 100 μl can be lost during filtration.

Hybridization Buffer: for 100 μl:

50%) Formamide 50 μl formamide

5X SSC 25 μl 20X SSC

0.1% SDS 25 μl 0.4% SDS

[0246] The solution was filtered through 0.2 μm syringe filter, then the volume was measured. About 1 μl of salmon sperm DNA (lOmg/ml) was added per 100 μl of buffer. Materials used for hybridization were: 2 Eppendorf tube racks, hybridization chambers (2 arrays per chamber), slides, coverslips, and parafilm. About 30 μl of nanopure water was added to each hybridization chamber. Slides and coverslips were cleaned using N₂ stream. About 30 μl of hybridization buffer was added to dried probe and vortexed-gently for 5 seconds. The probe remained in the dark for 10-15 minutes at room temperature and then was gently vortexed for several seconds and then was flash spun in the microfuge. The probes were boiled for 5 minutes and centrifuged for 3 min at 20800 x g (14000 rpm, Eppendorf model 5417C). Probes were placed in 70 °C heat block. Each probe remained in this heat block until it was ready for hybridization. [0247] Pipette 25 μl onto a coverslip. It is highly preferable to avoid the material at the bottom ofthe tube and to avoid generating air bubbles. This may mean leaving about 1 μl remaining in the pipette tip . The slide was gently lowered, face side down, onto the sample so that the coverslip covered that portion ofthe slide containing the array. Slides were placed in a hybridization chamber (2 per chamber). The lid ofthe chamber was wrapped with parafilm and the slides were placed in a 42°C humidity chamber in a 42°C incubator . It is preferable to not let probes or slides sit at room temperature for long periods. The slides were incubated for 18-24 hours.

Post-Hybridization Washing

[0248] To obtain single stranded cDNA probes on the array, all nonspecifically bound cDNA probe should be removed from the array. Removal of all non-specifically bound cDNA probe was accomplished by washing the array and using the following materials: slide holder, glass washing dish, SSC, SDS, and nanopure water. It is highly preferable that great caution be used with the standard wash conditions as deviations can greatly affect data. [0249] Six glass buffer chambers and glass slide holders were set up with 2X SSC buffer heated to 30-34°C and used to fill up glass dish to 3/4th of volume or enough to submerge the microarrays. It is important to exercise caution in heating ofthe 2X SSC buffer since a temperature of greater than 35°C might strip off the probes. The slides were removed from chamber and placed in glass slide holders. It is preferable that the slides are not allowed dry out. The slides were placed in 2X SSC buffer but it is recommended that no more than 4 slides be placed per dish. Coverslips should fall off within 2 to 4 minutes. In the event that the coverslips do not fall off within 2 to 4 minutes, very gentle agitation may be administered. The stainless steel slide carriers were placed in the second dish and filled with 2X SSC, 0.1% SDS. Then the slides were removed from glass slide holders and placed in the stainless steel holders submerged in 2X SSC, 0.1% SDS and soaked for 5 minutes. The slides were transferred in the stainless steel slide carrier into the next glass dish containing 0.1X SSC and 0.1% SDS for 5 minutes. Then the slides are transferred in the stainless steel carrier to the next glass dish containing only 0.1X SSC for 5 minutes. The slides, still in the slide carrier, was transferred into nanopure water (18 megaohms) for 1 minute. [0250] To dry the slides, the stainless steel slide carriers were placed on micro-carrier plates with a folded paper towel underneath. The top ofthe slides were gently dabbed with a tissue. Then the slides were spun in a centrifuge (Beckman GS-6 or equivalent) for 5 minutes at 1000 rpm. It is very important that the slides do not air dry, as this will lead to increased background.

EXAMPLE 15: USE OF ALGORITHMS TO IDENTIFY, SELECT, AND EVALUATE TOXICOLOGICALLY RELEVANT GENES

[0251] A two-step approach has been used in ranking candidate genes from the human array.

[0252] First, three cutoff criteria were specified for individual gene values from experiments involving expression analysis of human genes: The three cutoff criteria were: fold induction/repression level, average fluorescence ofthe replicate spots used to calculate expression level, and coefficient of variation ofthe replicate spots.

[0253] To calculate fold induction/repression level, the stored induction repression level of a gene in a particular experiment has been calculated as follows:

[0254] 1). arrive at a treatment score for the gene. The treatment score was represented by the amount of Cy3 labeled cDNA from a treated source (e.g., human or non-human cells or laboratory animals dosed with an agent) that had bound to a complementary target DNA spot ofthe microarray slide. The amount of Cy3 labeled cDNA was detected by a microarray laser scanner at a wavelength of 532nm.

[0255] 2). arrive at a control score for the gene. The control score was represented by the amount of Cy5 labeled cDNA from an untreated source that had bound to a complementary target DNA spot ofthe microarray slide. The amount of Cy5 labeled cDNA was detected by a microarray laser scanner at a wavelength of 635nm. The unit of measure was the pixel intensity or the average of several pixel intensities reported by a microarray laser scanner at coordinate on a microarray slide. The pixel intensity at that location was proportional to the number of photons detected by a photomultiplier tube when a spot oftarget DNA labeled with fluorescent probe was illuminated by a laser with a wavelength to which the dye is sensitive. For an Axon Instruments Inc. GenePix 4000A MicroArray Scanner, which was used in these experiments, these values are between 0 and 65535.

[0256] 3). compute an un-normalized freated-to-confrol ratio by dividing the treatment score by the control score

[0257] 4). compute a final, "normalized" induction score by dividing the un- normalized ratio by some normalization factor. For example, using the following 4-steps procedure above, calculation of Gene A is performed as follows: 1). Suppose that the average of its set of four raw treated values is 100,000 2). Suppose that the average ofthe set of its four raw control values is 25,000 3). The un-normalized ratio would thus be 4, and finally 4). If the normalization factor (designed to take into account environmental effects) turned out to be 2, one would end up with at a final, normalized induction score (often called fold induction) of 4 divided by 2, or 2. In this example, Gene A was induced two-fold. In all experiments conducted with respect to this invention, there were four replicates of each gene, with each replicate having a treated and a control value. Thus, the calculation of an expression level for each gene in an experiment involved aggregating eight data points.

[0258] The average fluorescence level ofthe replicate spots used to calculate the expression level is accomplished by a simple average ofthe four treated replicate values used in any experiment to calculate the expression level of a gene. [0259] The coefficient of variation ofthe replicate spots was a conventional measure of variability, expressed as a percentage, that in this case was derived by dividing the standard deviation ofthe four replicate treated-to-control ratios by the average ofthe four replicated treated-to-control ratios. The latter criteria represent useful measures of data quality. Thus, initial screening is based on expression level and measurement quality.

[0260] Algorithms were written specifically to perform the entire process of ranking genes to be included on the human toxicity array. In the initial part ofthe program, the three criteria above were "ended" together, so that in order to "make the first cut" the gene would have to meet all three criteria. This was considered the first tier in the program.

[0261] The criteria are adjustable within the algorithms. For the actual ranking ofthe human toxicity array, the following criteria were used: induction/repression level: 2; fluorescence level: 400; and a coefficient of variation: 30%. To make the first cut and be selected as a potential toxicologically relevant gene, a gene only had to meet the 3 criteria within one experiment. Likewise, relevant values for the gene would have been included each time it met the criteria within a different experiment. The data for genes that made the cut (and hence were selected as potential toxicologically relevant genes) and each time the genes made the cut were stored in a separate, temporary data table for ranking (the second tier in the process). A gene's data could be included more than once: one time for each experiment in which that gene met the three criteria.

[0262] Each ofthe factors listed in the table below was given a relative weight, as indicated:

[0263] Next, gene values that made the cut were aggregated into overall scores. It was necessary to "aggregate" the potentially more-than-one values for each gene into overall scores for the gene because data for a particular gene were included for each experiment in which that gene's values met the 3 cutoff criteria described above. Overall scores were aggregated for each six factors, as shown in the table above, and ranked for each gene, based on six ranking criteria: 1). Number of slides on which that gene met the cutoff criteria (NC = number of compounds). As noted, a gene could meet the 3 cutoff criteria described above in more than one ofthe experiments. NC is a simple count ofthe number of different compounds (not experiments, as duplicate experiments were performed for each compound) in which that gene met the 3 criteria, 2). Percent of consistency between slides (% time the gene value made the cutoff criteria on the replicated slide for that initial slide) (CC). As mentioned above, duplicate experiments were performed for each compound. CC is a count ofthe number of times the gene made the cutoff in both ofthe duplicate experiment pair. Thus, a gene could have anNC (number of compounds) of 3 but a CC (compound consistency) of 2, meaning that in two ofthe three compounds it made the cutoff in each ofthe duplicate experiments, and in one ofthe compounds, it did not. 3). Average magnitude (absolute value) of fold induction for all occurrences where that gene made the cutoff criteria (FI). This is a simple average ofthe magnitude ofthe expression/repression levels for each set of data values for a particular gene. 4). coefficient of variation of those fold induction scores (unlike all the other ranking criteria, a lower coefficient of variation is deemed better) (CV). The coefficient of variation applied to the set of expression values for a particular gene to assess the variability of its scores. 5). Average fluorescence value of all replicate spots of occurrences where that gene made the cutoff criteria (FL). This is a simple average ofthe fluorescence levels ofthe treated values for each occurrence of a gene that had made the cut. 6). Tissue consistency, i.e., what percent of cutoff-meeting occurrences ofthe gene were in the same tissue (CT). Expression levels were measured not only in several compounds but in several tissues as well (liver, kidney, etc.) CT was calculated as the percentage of time the gene data values which made the cutoff were measured in the same tissue. For example, if a gene had made the cut in four experiments, twice in liver and twice in kidney, its tissue consistency would be 50%.

[0264] The following weightings were used in the actual process of ranking genes for inclusion on the human array: NC=3, CC=1, Fl=5, CV=0, FL=0, CT=0. Thus, the final three criteria received no weight in this case. [0265] Each gene was assigned a score between 0 and 100 for each ranking criterion. Each ranking criterion score was computed as follows: The range of values for all genes was computed for the criterion by subtracting the lowest value present among all scores from the highest. The temporary data table compiled from the experiments included scores for every gene that had met the initial 3 cutoff criteria. A gene would be present in the table with values for each experiment in which it had met or surpassed the 3 cutoff criteria. Scores were then aggregated from the gene occurrences of each gene into an overall score for that gene for each ofthe six criteria described above. For example, if Gene A made the cut in three experiments with respective induction scores of 4, 6, and 8 then the aggregated induction score for Gene A would be 6 (the average ofthe three values). Further, for example, suppose the gene with the highest overall score on the induction factor had an overall score of 10, and the lowest an overall score of 2. Then, Gene A would receive a rating of 50%, because its score was halfway between the highest and the lowest. The score for each gene was then calculated by subtracting the lowest value present from the value for that gene, then dividing by the range and multiplying by 100. In other words, the score for each gene is the percent above the minimum present toward the maximum. For example, if a gene's score was three-fourths ofthe way between the minimum present and the maximum for that criterion, its score would be between the minimum present and the maximum for that criterion, its score would be 5%. Since for the CV factor (coefficient of variation of fold inductions) lower was deemed better, the score thus computed was subtracted from 100 to invert the percentage.

[0266] The final ranking score for each gene was computed via a weighted combination of its score on the six ranking criteria. If a score could not be computed for a particular criterion, the entire value of that criterion was removed from the equation , and ranking was based solely on the remaining factors. Each ofthe ranking criteria could be weighted between 0 and 5, and weightings are relative, so that 2:2:2:2:2:2 would be the same as 4:4:4:4:4:4, etc. A zero weighting would drop the factor from the equation.

[0267] For example, suppose that Gene A had the following scores: NC: 75%, CC: 50%, FI: 80%, CV: 25%, FL: 50%, and CT: 30%. Using the weightings described above (3, 1, 5, 0, 0, 0) the final score for Gene A would be calculated as follows:

[0268] (75*3 + 50* 1 + 80*5 + 25*0 + 50*0 + 30*0) / 9 (NOTE: 9 is the total number of "weightings" or 3+1+5)

[0269] (225 + 50 + 400 + 0 +0 + 0) 19

[0270] 675/9

[0271] 75 would be the final score for Gene A.

[0272] A score was then computed for each discrete gene based on aggregating the individual values for that gene from one or more experiments where that gene had made the initial cut. The list of genes was then rank-ordered on the basis of final scores. An objective determination was made of how many genes to take from the top ofthe list to be added to the human toxicity array. The human toxicity gene array and sequences is shown in Table 1.

Claims

1. A method for determining a toxicological response to an agent, the method comprising:

(a) exposing cells to an agent or isolating cells from a human subject who was exposed to an agent;

(b) obtaining a test expression profile of one or more human toxic response genes in the cells selected from the group consisting ofthe genes corresponding to the gene sequences in Tables 1, 2 or 5; and

(c) comparing the test expression profile to a reference gene expression profile of human toxic response genes indicative of toxicity, thereby to determine the presence of a toxic response to the agent.

2. The method of claim 1 , wherein the toxicity ofthe agent is evaluated by determining if there is a significant correlation between the test expression profile and the reference expression profile.

3. The method of claim 1 , wherein the toxicity of the agent is evaluated by determining if there is a pattern of expression that is predictive of specific toxicity endpoints as determined by predictive models.

4. The method of claim 1 , wherein the cells are derived from pancreas, thyroid, liver, lung, heart, kidney, spleen, testes, thymus, skin, bone, muscle, gastrointestinal tract, brain or nucleated cells present in the blood.

5. The method of claim 1 , wherein the reference gene expression profile indicative of toxicity is stored in a database.

6. The method of claim 1, wherein the cells are from an organ, tissue, or cell culture, and wherein the test expression profile of human toxic response genes is compared to the reference gene expression profile to determine the presence of a toxicological response in the organ, tissue or cell culture.

7. The method of claim 1 , wherein the agent is a pharmaceutical or industrial composition, and wherein the method comprises a method of screening the agent to determine a toxicological response ofthe pharmaceutical or industrial agent in the cells.

8. The method of claim 1 , wherein the cells exposed in step a) are cells obtained from a human tissue, blood, sweat, saliva, urine or fecal sample.

9. The method of claim 1 , wherein the agent is exposed to the cells at various concentrations or for various amounts of time or cells are derived from individuals exposed the agent at various concentrations by various routes of exposure for various amounts of time.

10. The method of claim 1 , wherein, in step b) the test expression profile of at least two human toxic response genes in the cells is obtained.

11. The method of claim 1 , wherein, in step b) the test expression profile of at least 10 human toxic response genes in the cells is obtained.

12. The method of claim 1, wherein, in step b) the test expression profile of at least 20 human toxic response genes in the cells is obtained.

13. The method of claim 1 , wherein, in step b) the test expression profile of at least 50 human toxic response genes in the cells is obtained.

14. The method of claim 1, wherein, in step b) the test expression profile of at least 200 human toxic response genes in the cells is obtained.

15. The method of claim 1 , wherein, in step b) the test expression profile of at least 500 human toxic response genes in the cells is obtained.

16. The method of claim 1 , wherein the gene expression profile is obtained using real-time polymerase chain reaction, Rnase protection, Northern blot, electrochemical hybridization detection, branched-chain to quantitatively detect levels of messenger RNA.

17. The method of claim 1 , wherein the gene expression profile is obtained by detecting protein expression.

18. An array comprising one or more polynucleotides selected from the group consisting ofthe genes corresponding to the partial sequences in Tables 1, 2, or 5, or fragments of at least 20 nucleotides thereof.

19. The array of claim 18, wherein genes corresponding to the partial gene sequences are responsive in cells derived from kidney, liver, spleen, heart, lung, testis, thymus, skin, bone, muscle, gastrointestinal tract, pancreas, thymus or brain.

20. The array of claim 18, wherein the array includes at least 25 of the polynucleotides.

21. The array of claim 18, wherein the array includes at least 50 ofthe polynucleotides.

22. The array of claim 18, wherein the array includes at least 200 of the polynucleotides.

23. The array of claim 18, wherein the array includes at least 500 of the polynucleotides.

24. The array of claim 18, wherein the polynucleotide fragment comprises at least 300 nucleotides.

25. An array comprising one or more polynucleotides which are homologous to the polynucleotides ofthe array of claim 18.

26. The method of claim 1, wherein the gene expression profile is obtained using an array of claim 18.

27. A method of determining if a gene putatively identified to be a toxic response gene plays a definite role in toxic response pathways by determining the expression profile of that gene after exposure of cells or a human subject to a known toxic pharmaceutical or industrial agent. The method comprising:

(a) exposing cells to an agent or isolating cells from a human subject who was exposed to an agent;

(b) obtaining the test gene expression profile for a putatively identified toxic response gene after exposure to a known toxic pharmaceutical or industrial agent c) comparing the test profile to the expression profile for a gene with a similar function or comparing the test profile to the expression profile of that gene after exposure to other known toxic compounds.

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Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

ATTAAACCTCTCGCCGAGCCCCTCCGCAGACTCTGCGCCGGAAAGTTTCATTTGCTG atcgcgggatccctctctggaatgat ttggccaagcttgagcccactatcc

TATGCCATCCTCGAGAGCTGTCTAGGTTAACGTTCGCACTCTGTGTATATAACCTCG gggtgca gagacacc

ACAGTCTTGGCACCTAACGTGCTGTGCGTAGCTGCTCCTTTGGTTGAATCCCCAGGC

CCTTGTTGGGGCACAAGGTGGCAGGATGTCTCAGTGGTACGAACTTCAGCAGCTTG

ACTCAAAATTCCTGGAGCAGGTTCACCAGCTTTATGATGACAGTTTTCCCATGGAA

ATCAGACAGTACCTGGCACAGTGGTTAGAAAAGCAAGACTGGGAGCACGCTGCCA

ATGATGTTTCATTTGCCACCATCCGTTTTCATGACCTCCTGTCACAGCTGGATGATC

AATATAGTCGCTTTTCTTTGGAGAATAACTTCTTGCTACAGCATAACATAAGGAAA

Interferon

AGCAAGCGTAATCTTCAGGATAATTTTCAGGAAGACCCAATCCAGATGTCTATGAT

H371A stimulator y gene CATTTACAGCTGTCTGAAGGAAGAAAGGAAAATTCTGGAAAACGCCCAGAGATTTA

ATCAGGCTCAGTCGGGGAATATTCAGAGCACAGTGATGTTAGACAAACAGAAAGA factor-3

GCTTGACAGTAAAGTCAGAAATGTGAAGGACAAGGTTATGTGTATAGAGCATGAA

ATCAAGAGCCTGGAAGATTTACAAGATGAATATGACTTCAAATGCAAAACCTTGCA

GAACAGAGAACACGAGACCAATGGTGTGGCAAAGAGTGATCAGAAACAAGAACA

GCTGTTACTCAAGAAGATGTATTTAATGCTTGACAATAAGAGAAAGGAAGTAGTTC

ACAAAATAATAGAGTTGCTGAATGTCACTGAACTTACCCAGAATGCCCTGATTAAT

GATGAACTAGTGGAGTGGAAGCGGAGACAGCAGAGCGCCTGTATTGGGGGGCCGC

CCAATGCTTGCTTGGATCAGCTGCAGAACTGGTTCACTATAGTTGCGGAGAGTCTG

CAGCAAGTTCGGCAGCAG

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NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

CCACTGGTACAAAATCTTTATGTAAGTATAAAATAAATAATATGAATATTAATTAA atcgcggaattcgaggccccatga ttggccaagcttggccttcctccag

TAACAACAACACAACAGCGGCAACAATATTAATAATAACAAGAGCTCTCCCATTGC atcaatgt ttttct

CCACGGCCTTCCTCCAGCTTTTCTCTTCTGCTTCACACAACTTTGTGAGATAGCTGTT

TTCATAGCTGGGAAAACTGAGGCCCAGAGAGGTCGGCACACTGGCCTGAGGTCAC

ACAGCAAGTCTGTAGGGCTGGGAGCCGAGGGAAGGGCAAAGGGGTCCCCCGGATC

CAGCCAGGGCAGAGAGGGGGATTTCAGGTTTAGGTCCAGAGGAGAGTGCCTCCCC

ACAGTCTGGCAGTCATTAGCAGGGTGATGGTGAGGCTGAGATGGGCAGGGCCAGG

CTGAGGAGCAGCCCCACTGCGGTACTGGACATCCAGGTCTGGGTGGTTACAGCCAC

TTTTAGTACAGCAGGAGACATCAATGTGGTTCATGCTGAAGGCGTCACCCAGGTGG

GCATGTTGGCACATTGAGGCGGTTGCACAGCCTCTTACCATATAGCTTTGGTTTTTC

GGTTCGTGAGTGCCGGTGGCTACCAGACATTGATTCATGGGGCCTCGGCAGTCAAT

GAGGAAAGTCTCTTCAGAGGAGCATCCATGGGTGCTGTTCCCCTTGCAGCTGTAAC

ACTGGCGGCCATTCTGCGGCAGATTTTCAAGCTCCAGGACTTCTTCACCTTCCTGGA

Urokinase

TCCAGTGGGTCACCACATCCAGGCACTGTTCTTCAGGGCTGCGGCACTGCAGGCTC

© plasminog

H402A en TGGTGCCGGCCCCTCTCACAGCTCATGTCTGATGAGCCACAGGAAATGCATTCGAG activator GTAACGGCTTCGGGAATAGGTGACAGCCCGGCCAGAGTTGCCCTGGTTGCACAAGT receptor CTAACCCACACACAACCTCGGTAAGGCTGGTGATCTTCAAGCCAGTCCGATAGCTC

AGGGTCCTGTTGGTCTTCTCTGAGTGGGTACAGCTTTTCTCCACCAGCTCCAGCTCT

TCTCCTTCTTCCCACAAGCGCACGATCGTGGTCCTGCAGAGGTCCTGTCCCAGGGCG

CACTCTTCCACACGGCAATCCCCGTTGGTCTTACACTGCATGCACCGCAGGCCCCAA

GAGGCTGGGACGCAGGTGTGGAGCAGCAGCAGCAGCGGCAGCAGCGGCGGGTGAC

CCATGTCGCGAGGGCAGCTCCTGTGCGCGGGGTCCCTGCACGTCTTCTCTCCTTCTG

GCCTCAGGAAGGAGGCAGTTTTGTGGCCCCAGGGACTCCTCCCAGACGTTTTGCGA

AAGAGCGAGTCAGCCCCAGATGCGTGGGCGCCTCCCCCTCCTCCCGTACGAACCTC

CTCCGCCACAAACTTCCTTCCCGGGGCGCGGCCGGCTGGTGGTGAAGGGGCTGGCT

CGGCCCTGACTCATGGAGTTGTGATCACAGCTCTGTCCCCAGATTGCCTGGGTGCA

AATCCCAGTTCTCTCTCTTCCTAACGTGGGACCCGGGGCAATCGCTCTCCACTGCTG

TAAAATGAGGATAAAAACTTTGACGGTAAATATGAATGTGGCTGATTATTTGGCTA

ACAGGATTTTTGTTAGTTTTGTCAGGAGGGATACTG

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NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

TCCACAGAAGATGTTTATTTGATGTAACAAAACAATACAGTTAGTQTTAGATCCAACCACAAAQAGCAAAAGGAATGAAAAATTTGTA

CAATGTACATAGAAAAAACAAGATATTTACTGTGACAACTATATATTCCTAAAATAATGCTTCTAAAATTACTCAATTATTGAAATCTAC cgc; gggatccggac ggccaagcttattgtgcaattgt

ATGAAAAAAAGGATGTTAATAGCGACAAAGTGCATAAAATCAAACATTGTATTTTGAGCAAATTAACATACTAGGCAATTTTGCTAAGA act Sgtggctcactac taccac

ATGCATGATTRRTTTTTTCTTGTTTACAGTCTGCTCAAAATATCTTTATACCAACAGGGTAGGCAGAACATTTAGGTTTAATATCAGTTAC ct

ACAATATRAGCATAAACTTCCACAACTACATAGGGTATTGTTTTCTTTTGAGCTGGCAAAGTGACTATAGAAACATCAGATGATTTCTCT

GAATTGAGAATTTTATCCAAATAAATGCCACATACCTTCTAGATATATGCATATCTTTCTATATTATGTAAATGGCTTTACCCATTTAAAT

AATAAACCATACAGCATTTAAGAATCATTATTATATGATTAACAATGTCATGTTCCAGGTTTAACAATTTCATAGGCCAAAAAAAATTTC

TTCTRAAAAACTAGTTTTATAAACGCAGAATATATTCCATGAGTAACTGCTGGTATTTTRTAAGAAAATATATTGTGCAATTGTGGCTAC

CACGTACTGCTGGCAAAGCATTATTATRTATGTAAAATGTGAAAAAAAAGGTGTAAAAATTTTTCAACTGCCTATGATCATGATGAAAG

GTTACTGCCTRCTTACAAAAATTATATTGGCATCTTCTTAAAAATAATTCGAAAAAGGGATAAACTCCCTAGCCAAAAATAAATAAATA

AAAAGGTGCATTTTTTAAAATGATGCTACTGCAATGCAATGGTTTAAATACCAAAAAACTGAGAAAATGAGCTGTCTGTGATCCAGCAT

TAAAGAACATACTAAAAAAGAGCATTAATGRAAATTAAGTAGAAAGGGGATCAAAAATTGAACTAACCAAGTTTGTGCAGTATTGTAG

CCAGGCTTCTAAAATTAGATGTAGAAAATATAAATAGACTGCTTTAGGTAGTGAGCCACCAGTGTCCAAAAAAAGGAATGAAATTAAG

AAAAAGCTCAGTTAACTTGATCCAAAGCTCTGAGTAATTCTTCACCCTGCAGTAGGTTTCTGCTGCCTTGTATAGGAGCATTAACTTCAC

AATCATAACTGGTCAGCTGTGGTAATCCACTTTCATCCATTGATTGCCCCAGCAGTCTACATGCTAAATCAGAGGGTATTAAAATAATTG

TCTTTTGCTCCATTCCATTCTGTTCACTAGATTTGCATCCTTRRACACGTTTCCAAGAAAGTGATGTAGTAGCTGCATGATCGTCTGGCTG

CTGTAATAATGTTCCAATTCCTACTGCTTGAAAAAGTGAACCATCATGTTCCATTTTTCGCTTTCTCTGAGCATTCTGCAAAGCTAGTATC

TTTGGATTTAGTTCTTCCTCAGGAACTGTAGTTCTTTGACTCAAAGCGACAGATAACACGTTAGGGCTTCTTGGATGAGATTTTTCTGTCT

Hypoxia- GTTCTATGACTCCTTTTCCTGCTCTGTTTGGTGAGGCTGTCCGACTTTGAGTATCTCTATATGGTGATGATGTGGCACTAGTAGTTTCTTT inducible ATGTATGTGGGTAGGAGATGGAGATGCAATCAATATTTTAATGTCTTCCATACGGTCTTTTGTCACTGTTTTTAATTCATCAGTGGTGGC κ> H325A AGTGGTAGTGGTGGCATTAGCAGTAGGTTCTTGTATTTGAGTCTGCTGGAATACTGTAACTGTGCTTTGAGGACTTGCGCTTTCAGGGCT factor 1 TGCGGAACTGCTTTCTAATGGTGACAACTGATCGAAGGAACGTAACTGGAAGTCATCATCCATTGGGATATAGGGAGCTAACATCTCCA alpha AGTCTAAATCTGTGTCCTGAGTAGAAAATGGGTTCTTTGCTTCTGTGTCTTCAGCAAAAAGTTTTTCTACCAATTCCAACTTGAATTCATT

GACCATATCACTATCCACATAAAAACAATATTCACTGGGACTATTAGGCTCAGGTGAACTTTGTCTAGTGCTTCCATCGGAAGGACTAG

GTGTCTGATCCTGAATCTGGGGCATGGTAAAAGAAAGTTCCAGTGACΤCTGGATTTGGTTCTAATTTTAATGCAACTTCTTGATTGAGTG

CAGGGTCAGCACTACTTCGAAGTGGCTTTGGCGTTTCAGCGGTGGGTAATGGAGACATTGCCAAATTTATATTCTGTAATTTTTCGTTGG

GTGAGGGGAGCATTACATCATTATATAATGGTACTTCCTCAAGTTGCTGGTCATCAGTTTCTGTGTCGTTGCTGCCAAAATCTAAAGATA

TGATTGTGTCTCCAGCGGCTGGGGCCAGCAAAGTTAAAGCATCAGGTTCCTTCTTAAGTTTGTCAAAGAGGCTACTTGTATCTTCTGATT

CAACTTTGGTGAATAGCTGAGTCATTTTCATATCTGAAGATTCAACCGGTTTAAGGACACATTCTGTTTGTTGAAGGGAGAAAATCAAGT

CGTGCTGAATAATACCACTCACAACGTAATTCACACATACAATGCACTGTGGTTGAGAATTCTTGGTGTTATATATGACAGTTGCTTGAG

TTTCAACCCAGACATATCCACCTCTTTTGGCAAGCATCCTGTACTGTCCTGTGGTGACTTGTCCTTTAGTAAACATATCATGATGAGTTTT

GGTCAGATGATCAGAGTCCAAAGCATGATAATATTCATAAATTGAGCGGCCTAAAAGTTCTTCTGGCTCATATCCCATCAATTCGGTAA

TTCTTTCATCACAATAAGAAAATTTCATATCCAGGCTGTGTCGACTGAGGAAAGTCTTGCTATCTAAAGGAATTTCAATATTTGATGGGT

GAGGAATGGGTTCACAAATCAGCACCAAGCAGGTCATAGGTGGTTTCTTATACCCACACTGAGGTTGGTTACTGTTGGTATCATATACG

TGAATGTGGCCTGTGCAGTGCAATACCTTCCATGTTGCAGACTTTATGTTCATAGTTCTTCCTCGGCTAGTTAGGGTACACTTCATTCTGA

GAAAAAAGCT CGCTGTGTGTTTTGTTCTTTACCCTTTTTCACAAGGCCATTTCTGTGTGTAAGCATTTCTCTCATTTCCTCATGGTCACAT

GGATGAGTAAAATCAAACACACTGTGTCCAGTTAGTTCAAACTGAGTTAATCCCATGTATTTGTTCACATTATCAGAAATGTAAATCATG

TCACCATCATCTGTGAGAACCATAACAAAACCATCCAAGGCTTTCAAATAAAAGCAATTCATCTGTGCTTTCATGTCATCTTCAATATCC

AAATCACCAGCATCCAGAAGTTΓCCTCACACGCAAATAGCTGATGGTAAGCCTCATCACAGAGGCCTTATCAAGATGCGAACTCACATT

ATGTGGAAGTGGCAACTGATGAGCAAGCTCATAAAAAACTTCAGATTCTTTACTTCGCCGAGATCTGGCTGCATCTCGAGACTTTTCTTT

TCGACGTTCAGAACTTATCTTTTTCTTGTCGTTCGCGCCGCCGGCGCCCTCCATGGTGAATCGGTCCCCGCGATGTCTTCAC

Ul

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Ul κ>

Ul Ul

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

ACGCCGCGCTCAGCTTCCATCGCTGGGCGGTCAACAAGTGCGGGCCTGGCTCAGCG cgcgggatccccagttgtccaaga ggccaagcttcctggcagatgttct

CGGGGGGGCGCGGAGACCGCGAGGCGACCGGGAGCGGCTGGGTTCCCGGCTGCGC cctggc gctcc

GCCCTTCGGCCAGGCCGGGAGCCGCGCCAGTCGGAGCCCCCGGCCCAGCGTGGTCC

GCCTCCCTCTCGGCGTCCACCTGCCCGGAGTACTGCCAGCGGGCATGACCGACCCA

CCAGGGGCGCCGCCGCCGGCGCTCGCAGGCCGCGGATGAAGAAGAAAACCCGGCG

CCGCTCGACCCGGAGCGAGGAGTTGACCCGGAGCGAGGAGTTGACCCTGAGTGAG

GAAGCGACCTGGAGTGAAGAGGCGACCCAGAGTGAGGAGGCGACCCAGGGCGAA

GAGATGAATCGGAGCCAGGAGGTGACCCGGGACGAGGAGTCGACCCGGAGCGAGG

AGGTGACCAGGGAGGAAATGGCGGCAGCTGGGCTCACCGTGACTGTCACCCACAG

H118C BAG-1 CAATGAGAAGCACGACCTTCATGTTACCTCCCAGCAGGGCAGCAGTGAACCAGTTG

TCCAAGACCTGGCCCAGGTTGTTGAAGAGGTCATAGGGGTTCCACAGTCTTTTCAG

AAACTCATATTTAAGGGAAAATCTCTGAAGGAAATGGAAACACCGTTGTCAGCACT

TGGAATACAAGATGGTTGCCGGGTCATGTTAATTGGGAAAAAGAACAGTCCACAG

GAAGAGGTTGAACTAAAGAAGTTGAAACATTTGGAGAAGTCTGTGGAGAAGATAG

CTGACCAGCTGGAAGAGTTGAATAAAGAGCTTACTGGAATCCAGCAGGGTTTTCTG

CCCAAGGATTTGCAAGCTGAAGCTCTCTGCAAACTTGATAGGAGAGTAAAAGCCAC

AATAGAGCAGTTTATGAAGATCTTGGAGGAGATTGACACACTGATCCTGCCAGAAA

ATTTCAAAGACAGTAGATTGAAAAGGAAAGGCTTGGTAAAAAAGGTTCAGGCATT

CCTAGCCGAGTGTGACACAGTGGAGC

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. sequence sequence

ATATGTGGCCAAATCATTCCTTGGAATTTCCCGTTGGTTATGCTCATTTGGAAGATA cgcgggatccagaggcagggtttc ggccaagctttagcctttattccccc

GGGCCTGCACTGAGCTGTGGAAACACAGTGGTTGTCAAACCAGCAGAGCAAACTCC ctcctg acgg

TCTCACTGCTCTCCACGTGGCATCTTTAATAAAAGAGGCAGGGTTTCCTCCTGGAGT

AGTGAATATTGTTCCTGGTTATGGGCCTACAGCAGGGGCAGCCATTTCTTCTCACAT

GGATATAGACAAAGTAGCCTTCACAGGATCAACAGAGGTTGGCAAGTTGATCAAA

GAAGCTGCCGGGAAAAGCAATCTGAAGAGGGTGACCCTGGAGCTTGGAGGAAAGA

GCCCTTGCATTGTGTTAGCTGATGCCGACTTGGACAATGCTGTTGAATTTGCACACC

ATGGGGTATTCTACCACCAGGGCCAGTGTTGTATAGCCGCATCCAGGATTTTTGTGG

Aldehyde AAGAATCAATTTATGATGAGTTTGTTCGAAGGAGTGTTGAGCGGGCTAAGAAGTAT

H123D dehydroge ATCCTTGGAAATCCTCTGACCCCAGGAGTCACTCAAGGCCCTCAGATTGACAAGGA nase 1 ACAATATGATAAAATACTTGACCTCATTGAGAGTGGGAAGAAAGAAGGGGCCAAA

CTGGAATGTGGAGGAGGCCCGTGGGGGAATAAAGGCTACTTTGTCCAGCCCACAGT

GTTCTCTAATGTTACAGATGAGATGCGCATTGCCAAAGAGGAGATTTTTGGACCAG

TGCAGCAAATCATGAAGTTTAAATCTTTAGATGACGTGATCAAAAGAGCAAACAAT

Ul Ul ACTTTCTATGGCTTATCAGCAGGAGTGTTTACCAAAGACATTGATAAAGCCATAAC

AATCTCCTCTGCTCTGCAGGCAGGAACAGTGTGGGTGAATTGCTATGGCGTGGTAA

GTGCCCAGTGCCCCTTTGGTGGATTCAAGATGTCTGGAAATGGAAGAGAACTGGGA

GAGTACGGTTTCCATGAATATACAGAGGTCAAAACAGTCACAGTGAAAATCTCTCA

GAAGAACTCATAAA

Ul oe

Table 1

NumeriArray

Sequence Left PCR primer Right PCR primer cal ref. name sequence sequence

GGGCGTGATTTGAGCCCCGTTTTTATTTTCTGTGAGCCACGTCCTCCTCGAGGGGGT cgcgggatccggcggaggaaaac ggccaagcttgtagcgaatgtcac

CAATCTGGCCAAAAGGAGTGATGCGCTTCGCCTGGACCGTGCTCCTGCTCGGGCCT tgtctgg gcgca

TTGCAGCTCTGCGCGCTAGTGCACTGCGCCCCTCCCGCCGCCGGCCAACAGCAGCC

CCCGCGCGAGCCGCCGGCGGCTCCGGGCGCCTGGCGCCAGCAGATCCAATGGGAG

AACAACGGGCAGGTGTTCAGCTTGCTGAGCCTGGGCTCACAGTACCAGCCTCAGCG

CCGCCGGGACCCGGGCGCCGCCGTCCCTGGTGCAGCCAACGCCTCCGCCCAGCAGC

CCCGCACTCCGATCCTGCTGATCCGCGACAACCGCACCGCCGCGGCGCGAACGCGG

ACGGCCGGCTCATCTGGAGTCACCGCTGGCCGCCCCAGGCCCACCGCCCGTCACTG

GTTCCAAGCTGGCTACTCGACATCTAGAGCCCGCGAACGTGGCGCCTCGCGCGCGG

Lysyl

H153B AGAACCAGACAGCGCCGGGAGAAGTTCCTGCGCTCAGTAACCTGCGGCCGCCCAG oxidase

CCGCGTGGACGGCATGGTGGGCGACGACCCTTACAACCCCTACAAGTACTCTGACG

ACAACCCTTATTACAACTACTACGATACTTATGAAAGGCCCAGACCTGGGGGCAGG

TACCGGCCCGGATACGGCACTGGCTACTTCCAGTACGGTCTCCCAGACCTGGTGGC

CGACCCCTACTACATCCAGGCGTCCACGTACGTGCAGAAGATGTCCATGTACAACC

TGAGATGCGCGGCGGAGGAAAACTGTCTGGCCAGTACAGCATACAGGGCAGATGT

CAGAGATTATGATCACAGGGTGCTGCTCAGATTTCCCCAAAGAGTGAAAAACCAAG

GGACATCAGATTTCTTACCCAGCCGACCAAGATATTCCTGGGAATGGCACAGTTGT

CATCAACATTACCACAGTATGGATGAGTTTAGCCACTATGACCTGCTTGATGCCAA

CACCCAGAGGAGAGTGGC

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CTGTTGCCATTATGTTTGCTTTATTGCCAAGATTGAATACAACTCTTTAATAAAATATATGCAATATGGTA cgcgggatcccggaaatggcaacc ggccctgcagacatcagcatct

AGATGAGCAAAATGAGATTTTCCCTAGTTCAACAGATAGAATTGAAGTAACAATCAATTTAATTAGGCAA

ACAGGGCTTGCCAATTAGAATGCAGGATTCCCTGGAGCCTTTTAAAACACAATGTAGAGAAAGTTGTTCT aatcat cgtgg

ATCTAGCCCAATATTTACAGTTGTGGAAGTTACACATGTAATTACAACATAAATATTCATCAAGATTTCTT

GAGTGAAACTGGGTACAAGTGAAATAAAGGAAAGTTATACATCAGATTAATATGAGAGAGAAAAAGGAG

CTAGACCCCTCCCCTGTAATCCCCATCACTGCACGTCCCAGATTTCACAGAGAAGTGGGGTAAACTTGTGG

TCGTTTACTCTCCATGACATCAGCATCTCAGCGTGGTGATGATTGAATGTCCGTAATTCAGTCAGGCGACC

CAGGAGACAGGCAAAGTGTTGAGGATTTTCAGGCTGGTGAATCTTACACAACTTTTGTAGCACATCAAGA

AGTGGCTCCTGAAGCTTCTCTACTGCCTCTCTATCCTTTATGTATTGTCTATCTGGAGACAGGATAACAATT

GCTGTAAGCAGAGCATACTCCTCTTQAGTCATTTTCAGTTCCCCAATACTTTTATAAAAACTAAACATAGG

TGTTATATATTCATCAGAGATACCACTATTTCGAATTCTTTCTTCCAATAGGTCAGAATGCCCAGACGGAA

GTTTCTTATTGAAAATCTCAGCTGAACGAAGGAACATAGCTTCAACCGCAGACCCTTTCAGCAAAGCAAT

CTGGTCTTCATGGTCCAAAGTCTGAAATCCTGGTAGCTTTTTTGTGAATTCTACAAGAACCTGTACATGAT

TGGTTGCCATTTCCGTCAAAATGAGAAAATTTTCTTCTGCACTGAATTCTTCTTTTAAAATTTTATTTGTTA

TTTCCTGAGGCATCCTCTGTTTGTTATATGAATCCATAATAAAATGTAGAAGAGTCTGTTGATCTGGGGTG

Farnesol AGTTCAGTTTTCTCCCTGCATGACTTTGTTGTCGAGGTCACTTGTCGCAAGTCACGACCTTCACTGTCTTCA

H155A receptor TTCACGGTCTGATCTGCATGCTGCTTCACATTTTTTCTCAGTCGCTTAGATTTACACTGAATTTCAGTTAAC

AAGCATTCAGCCAACATTCCCATCTCTTTGCATTTCCTTAGTCGACACTCTTGACACTTTCTTCGCATGTAC

ATATCCATCACACAGTTGCCCCCGTTTTTACACTTGTACACAGCGTTTTTGGTAATGCTTCTCCTGAAGAAA

CCTTTACACCCCTCACAGGTCAGTGCATTATAGTGGTATCCAGAGGCTCTGTCTCCACAAACAACACACAG

CTCATCCCCTTTGATCCTCCCTGCTGACGCGCCCATGCGGGGCTTCTTTGTTACAGGCATCTCTGCTACCTC

AGTTTCTCCCTGGTAGAGAGTCTCAGCTGGCATACGCCTGAGTTCATATATTCCAGGAGAGTACCACTCTT

CAGGCTGCTGGGGGTAGAAACCCAGGTTGGAATAATAGGATGACGAGGAAATCTGTGGTTGAACTTGGG

GAAACTGAACATTGCTGTATTGCGAGTATGGTTCCACTTCCAGGTTCTGTCCCAGAGGACCTGCCACTTGT

TCTGTTAAAACACCAAATAAATTTTCAGAAAAAGAAAATTCATCTGTGGTAGGTAAATGGGAATGTTCAA

TGAGATTCATTTTTGATCCCATCCAAATTTTTCAATTGAAATGCACTTTCTTTATGGTGGTCTTCAAAAAAA

ACTCCAAAGTGTCTGAAGTTTCATCTTGAGGAAATGTCCAGAAGAAATCCAGGAAACTAAGAGAAGCAGT

GTTCACTTTGAGCTATGTTTCTAAGTCTTCTTTTCTTCTTTCACTCCTTCTACGATGTCTTCTACCTCCTTGG

ATTGTTTTGGGTCAGAGATGGACTTTCAAGGCCCTGGGAGGATTCTGGACTGAGTCTTCCTCTCCAGATCC

CAGCGATTTTGCTACAAATGCTCAGAATCCAATTTCGCATTAGGATAAGTCGGGGAGACAATGAGGTGAG

GAGGAGGAGAGAGTCTCGT

Table 1

I NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

TCTTGCTGCGCCTCCGCCTCCTCCTCTGCTCCGCCACCGGCTTCCTCCTCCTGAGCAG cgcgggatcccaggactgaacgtc ggccctgcagtgacacaaacatg

TCAGCCCGCGCGCCGGCCGGCTCCGTTATGGCGACCCGCAGCCCTGGCGTCGTGAT ttgctcg tcaaatccc

TAGTGATGATGAACCAGGTTATGACCTTGATTTATTTTGCATACCTAATCATTATGC

TGAGGATTTGGAAAGGGTGTTTATTCCTCATGGACTAATTATGGACAGGACTGAAC

GTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTGTAGCCCTCTGT

GTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTGGATTACATCAAAGCACT

Hypoxant GAATAGAAATAGTGATAGATCCATTCCTATGACTGTAGATTTTATCAGACTGAAGA hine- GCTATTGTAATGACCAGTCAACAGGGGACATAAAAGTAATTGGTGGAGATGATCTC

TCAACTTTAACTGGAAAGAATGTCTTGATTGTGGAAGATATAATTGACACTGGCAA

H193D guanine phosphori AACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGG bosyltrans TCGCAAGCTTGCTGGTGAAAAGGACCCCACGAAGTGTTGGATATAAGCCAGACTTT ferase GTTGGATTTGAAATTCCAGACAAGTTTGTTGTAGGATATGCCCTTGACTATAATGAA

TACTTCAGGGATTTGAATCATGTTTGTGTCATTAGTGAAACTGGAAAAGCAAAATA

CAAAGCCTAAGATGAGAGTTCAAGTTGAGTTTGGAAACATCTGGAGTCCTATTGAC

ATCGCCAGTAAAATTATCAATGTTCTAGTTCTGTGGCCATCTGCTTAGTAGAGCTTT

TTGCATGTATCTTCTAAGAATTTTATCTGTTTTGTACTTTAGAAATGTCAGTTGCTGC

ATTCCTAAACTGTTTATTTGCACTATGAGCCTATAGACTATCAGTTCCCTTTGGGCG

GATTGTTGTTTAACTTGTAAATGAAAAAATTCTCTTAAACCACAGCACTATTGAGTG

AAACATT

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GGTCCACCATGGCCCTGCTGCACTCCGGCCGCGTCCTCCCCGGGATCGCCGCCGCCT cgcgggatccaaccttatcaacagg ggccaagcttgcagcacgcaagc

TCCACCCGGGCCTCGCCGCCGCGGCCTCTGCCAGAGCCAGCTCCTGGTGGACCCAT ccgca tagaat

GTGGAAATGGGACCTCCAGATCCCATTCTGGGAGTCACTGAAGCCTTTAAGAGGGA

CACCAATAGCAAAAAGATGAATCTGGGAGTTGGTGCCTACCGGGATGATAATGGA

AAGCCTTACGTTCTGCCTAGCGTCCGCAAGGCAGAGGCCCAGATTGCCGCAAAAAA

TTTGGACAAGGAATACCTGCCCATTGGGGGACTGGCTGAATTTTGCAAGGCATCTG

CAGAACTAGCCCTGGGTGAGAACAGCGAAGTCTTGAAGAGTGGCCGGTTTGTCACT

Aspartate GTGCAGACCATTTCTGGAACTGGAGCCTTAAGGATCGGAGCCAGTTTTCTGCAAAG aminotran ATTTTTTAAGTTCAGCCGAGATGTCTTTCTGCCCAAACCAACCTGGGGAAACCACAC

H195B sferase, ACCCATCTTCAGGGATGCTGGCATGCAGCTACAAGGTTATCGGTATTATGACCCCA mitochond AGACTTGCGGTTTTGACTTCACAGGCGCTGTGGAGGATATTTCAAAAATACCAGAG rial CAGAGTGTTCTTCTTCTGCATGCCTGCGCCCACAATCCCACGGGAGTGGACCCGCGT

CCGGAACAGTGGAAGGAAATAGCAACAGTGGTGAAGAAAAGGAATCTCTTTGCGT

TCTTTGACATGGCCTACCAAGGCTTTGCCAGTGGTGATGGTGATAAGGATGCCTGG

GCTGTGCGCCACTTCATCGAACAGGGCATTAATGTTTGCCTCTGCCAATCATATGCC

AAGAACATGGGCTTATATGGTGAGCGTGTAGGAGCCTTCACTATGGTCTGCAAAGA

TGCGGATGAAGCCAAAAGGGTAGAGTCACAGTTGAAGATCTTGATCCGTCCCATGT

ATTCCAACCCTCCCCTCAATGGGGCCCGGATTGCTGCTGCCATTCTGAACACCCCAG

ATTTGCGAAAACA

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CCATCCAGTGCCTTCCGCAGCGCCGCTAAAGCGCAGTTCTCGTTGGTGTAACTTTTT cgcgggatcctttcgaaaccatcatt ggccaagcttcttctgtgccacact

CTTTTTTTTTTCAGCCACTTCCGGCTCCTGCGTCGCTCCGGAAGCCTGCGAGTTCCG tagcatca aggacaag

GAAGCCTTGGTAATCCAGATTCGGCTAGGAAAAGACAAGCTTTCCAGAGAATGTTT

CAGAGAAAGTTACGTGGAGCGTGGGCGTTTCGCAGACTCCTAAGTGGTCTGGAACC

TAACCTGCAGTGTCTCTGAGCTTCTGGCAAGTATGAGGCAAGGATTTCACAGAAGA

ACTTGGAGCAAAAATTCCCACTGTCATAGCTATAGGCTGTCCAGTCTTGAGGGAGC

TGCAGGAACAGGAAAAAGAGACCTTCAGAGAAGACATGGCTTCCAGAAAAGAGAA

TGCGAAGAGTGCAAACAGAGTGCTAAGAATAAGCCAGTTGGATGCACTTGAACTA

Peroxisom

AACAAGGCCCTGGAGCAGCTAGTTTGGTCCCAGTTTACTCAGTGCTTTCATGGATTT e

H202F AAACCTGGGCTGTTAGCTCGCTTTGAGCCAGAGGTGAAAGCGTGCTTATGGGTTTT assembly factor 1 CTTGTGGAGATTCACCATCTACTCCAAAAATGCCACAGTGGGACAGTCAGTTTTGA

ATATTAAGTACAAAAATGATTTTTCCCCTAACCTGAGATATCAGCCACCCAGTAAA

AATCAAAAAATCTGGTATGCTGTTTGTACAATTGGTGGCAGGTGGTTAGAAGAACG

ATGCTATGATTTGTTTCGAAACCATCATTTAGCATCATTTGGGAAAGTCAAGCAGTG

TGTGAATTTTGTGATTGGACTTTTGAAATTAGGTGGGCTGATTAATTTTTTGATTTTC

CTTCAGAGGGGAAAGTTTGCAACTTTGACAGAACGTCTCCTAGGTATTCATTCTGTA

TTTTGCAAGCCTCAAAACATACGTGAAGTTGGCTTTGAATACATGAATAGGGAACT

TCTCTGGCATGGTTTTGCTGAATTTCTGATTTTTCTCTTACCACTTATCAATGTCCAG

AAGTTGAAA

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

TGTGCTCGCTGCTCAGCGCGCACCCGGAAGATGAGGCTCGCCGTGGGAGCCCTGCT cgcgggatccagctgtgtatgggct ggccctgcagcaggttaccaaca

GGTCTGCGCCGTCCTGGGGCTGTGTCTGGCTGTCCCTGATAAAACTGTGAGATGGT caggc ccttgaca

GTGCAGTGTCGGAGCATGAGGCCACTAAGTGCCAGAGTTTCCGCGACCATATGAAA

AGCGTCATTCCATCCGATGGTCCCAGTGTTGCTTGTGTGAAGAAAGCCTCCTACCTT

GATTGCATCAGGGCCATTGCGGCAAACGAAGCGGATGCTGTGACACTGGATGCAG

GTTTGGTGTATGATGCTTACTTGGCTCCCAATAACCTGAAGCCTGTGGTGGCAGAGT

TCTATGGGTCAAAAGAGGATCCACAGACTTTCTATTATGCTGTTGCTGTGGTGAAG

AAGGATAGTGGCTTCCAGATGAACCAGCTTCGAGGCAAGAAGTCCTGCCACACGG

GTCTAGGCAGGTCCGCTGGGTGGAACATCCCCATAGGCTTACTTTACTGTGACTTAC

H205B Transferri n CTGAGCCACGTAAACCTCTTGAGAAAGCAGTGGCCAATTTCTTCTCGGGCAGCTGT

GCCCCTTGTGCGGATGGGACGGACTTCCCCCAGCTGTGTCAACTGTGTCCAGGGTG

TGGCTGCTCCACCCTTAACCAATACTTCGGCTACTCGGGAGCCTTCAAGTGTCTGAA

GGATGGTGCTGGGGATGTGGCCTTTGTCAAGCACTCGACTATATTTGAGAACTTGG

CAAACAAGGCTGACAGGGACCAGTATGAGCTGCTTTGCCTAGACAACACCCGGAA

Ul

© GCCGGTAGATGAATACAAGGACTGCCACTTGGCCCAGGTCCCTTCTCATACCGTCG

TGGCCCGAAGTATGGGCGGCAAGGAGGACTTGATCTGGGAGCTTCTCAACCAGGCC

CAGGAACATTTTGGCAAAGACAAATCAAAAGAATTCCAACTATTCAGCTCTCCTCA

TGGGAAGGACCTGCTGTTTAAGGACTCTGCCCACGGGTTTTTAAAAGTCCCCCCAA

GGATGGATGCCAAGA

Table 1

NumeriArray Sequence Left PCR primer Right PCR primer cal ref. name sequence sequence

GAGGAGCAGCGAGTCAAGATGAGAGTTCAGCCGCGGCGGCAGCAGCAGCAGACTC cgcgggatcccagccaggtgatgg ggccaagcttgagagttctggatg

AAGAATGAACAATCCGTCAGAAACCAGTAAACCATCTATGGAGAGTGGAGATGGC tgacag actcagca

AACACAGGCACACAAACCAATGGTCTGGACTTTCAGAAGCAGCCTGTGCCTGTAGG

AGGAGCAATCTCAACAGCCCAGGCGCAGGCTTTCCTTGGACATCTCCATCAGGTCC

AACTCGCTGGAACAAGTTTACAGGCTGCTGCTCAGTCTTTAAATGTACAGTCTAAAT

CTAATGAAGAATCGGGGGATTCGCAGCAGCCAAGCCAGCCTTCCCAGCAGCCTTCA

GTGCAGGCAGCCATTCCCCAGACCCAGCTTATGCTAGCTGGAGGACAGATAACTGG

GCTTACTTTGACGCCTGCCCAGCAACAGTTACTACTCCAGCAGGCACAGGCACAGG

Octamer- CACAGCTGCTGGCTGCTGCAGTGCAGCAGCACTCCGCCAGCCAGCAGCACAGTGCT

H207A binding GCTGGAGCCACCATCTCCGCCTCTGCTGCCACGCCCATGACGCAGATCCCCCTGTCT protein 1 CAGCCCATACAGATCGCACAGGATCTTCAACAACTGCAACAGCTTCAACAGCAGAA

TCTCAACCTGCAACAGTTTGTGTTGGTGCATCCAACCACCAATTTGCAGCCAGCGCA

GTTTATCATCTCACAGACGCCCCAGGGCCAGCAGGGTCTCCTGCAAGCGCAAAATC

TTCAAACGCAACTACCTCAGCAAAGCCAAGCCAACCTCCTACAGTCGCAGCCAAGC

ATCACCCTCACCTCCCAGCCAGCAACCCCAACACGCACAATAGCAGCAACCCCAAT

TCAGACACTTCCACAGAGCCAGTCAACACCAAAGCGAATTGATACTCCCAGCTTGG

AGGAGCCCAGTGACCTTGAGGAGCTTGAGCAGTTTGCCAAGACCTTCAAACAAAGA

CGAATCAAACTTGGATTCACTCAGGGTGATGTTGGGCTCGCTATGGGGAAACTATA

TGGAAATGACTTCAG

Ul

ON

Table 1

NumeriArray

Sequence Left PCR primer Right PCR primer cal ref. name sequence sequence

TTCGGGGTTCCCTCCCAGGATCGACATGCACTTGGATCATTGCAGTTTCTGGGCCTG cgcgggatccccatcttaactgagg ggccctcgagtgaaaagcactgct

CTGTCATCTCCCCCTTCAGTACTTCCCACATATGACTACCAACTGTTAGACGATGTG ctcaggc ccaacctt

CTGCTGCCAGACCAGCCTTTCATTTCTTGGACATCCGGCTATTTACCCAGGAAAAGT

CATCCTTTGACTATTTTTCATGACCTTTCTCATCTGACTCGTGATCTGCCCATCTTGT

TACTCCCTAGCAGTGTGGTAAGAGGTCAACCAGAATCAAGATGTCTGGGACTGAGG

AAGCAATTCTTGGAGGACGTGACAGCCATCCTGCTGCTGGCGGCGGCTCAGTGTTA

DNA TGCTTTGGACAGTGCCAGTACACAGCAGAAGAGTACCAGGCCATCCAGAAGGCCCT repair and GAGGCAGAGGCTGGGCCCAGAATACATAAGTAGCCGCATGGCTGGCGGAGGCCAG recombina AAGGTGTGCTACATTGAGGGTCATCGGGTAATTAATCTGGCCAATGAGATGTTTGG

H221G tion TTACAATGGCTGGGCACACTCCATCACGCAGCAGAATGTGGATTTTGTTGACCTCA homologu ACAATGGCAAGTTCTACGTGGGAGTCTGTGCATTTGTGAGGGTCCAGCTGAAGGAT e (RAD GGTTCATATCATGAAGATGTTGGTTATGGTGTTAGTGAGGGCCTCAAGTCCAAGGC

52) TTTATCTTTGGAGAAGGCAAGGAAGGAGGCGGTGACAGACGGGCTGAAGCGAGCC

CTCAGGAGTTTTGGGAATGCACTTGGAAACTGTATTCTGGACAAAGACTACCTGAG

Ul -4 ATCACTAAATAAGCTTCCACGCCAGTTGCCTCTTGAAGTGGATTTAACTAAAGCGA

AGAGACAAGATCTTGAACCGTCTGTGGAGGAGGCAAGATACAACAGCTGCCGACC

GAACATGGCCCTGGGACACCCACAGCTGCAGCAGGTGACCTCCCCTTCCAGACCCA

GCCATGCTGTGATACCGGCGGACCAGGACTGCAGCTCCCGAAGCCTGAGCTCATCC

GCCGTGGAGAGCGA

Ul oe

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

ACCCAAGCCAGCACTGGCTCATACTGATTCATTTTGATCTCTGCTAATACCAGAGTC cgcggaattcgatggccaggcctc ggccaagcttttcaaagggagatg

CTGCGTGGCAGAGCCATTGGCACCAGAAATTACAAGAGAACATGGCCAAGCGAGT actgta agactcttg

TGCCATTGTGGGAGCTGGGGTCAGCGGCCTGGCCTCCATCAAGTGCTGTCTGGAAG

AAGGACTGGAGCCCACCTGCTTTGAGAGGAGCGATGACCTTGGGGGGCTGTGGAG

ATTCACCGAACATGTTGAAGAAGGCAGAGCCAGTCTCTACAAGTCTGTGGTTTCCA

ACAGCTGCAAGGAGATGTCTTGTTACTCAGACTTTCCATTCCCAGAAGATTATCCAA

ACTATGTGCCAAATTCTCAATTCCTGGAATATCTCAAAATGTATGCAAACCACTTTG

Flavin ACCTTCTGAAACACATTCAATTCAAGACCAAAGTCTGCAGTGTAACAAAATGCTCA containing GATTCTGCTGTCTCTGGCCAATGGGAGGTGGTCACTATGCATGAAGAGAAGCAAGA

H223B GTCAGCCATCTTTGATGCTGTCATGGTCTGCACTGGCTTTCTTACTAATCCTTATTTG monooxyg enase 1 CCACTGGATTCCTTTCCAGGTATTAATGCCTTTAAAGGCCAGTACTTTCATAGCCGG

CAATATAAGCATCCAGATATATTTAAGGACAAGAGAGTCCTTGTGATTGGAATGGG

AAATTCTGGCACAGACATTGCTGTGGAGGCCAGCCACCTGGCGGAAAAGGTGTTCC

TCAGCACCACCGGAGGGGGATGGGTGATCAGCCGAATCTTTGACTCGGGCTACCCA

TGGGACATGGTGTTCATGACACGCTTTCAGAACATGTTGAGAAATTCCCTCCCAAC

CCCAATTGTGACTTGGTTGATGGAGCGAAAGATAAACAACTGGCTCAATCATGCAA

ATTACGGCTTAATACCAGAAGACAGGACTCAGCTGAAAGAGTTTGTGCTAAATGAT

GAGCTCCCAGGACGCATCATCACTGGGAAAGTGTTCATCAGGCCAAGCATAAAAG

AGGTAAAGGAAAA

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

CTGGCAGGCAGGACTGGGATCGAGGCCCAGAAAACGGAGCAGCGGGCACCAGGGA cgcgggatcccctgctcaggctgat ggccaagcttggacgaatgcaaa

GGCCTGGAACGGGGCGAGCGCCATGAGCAACAAATGCGACGTGGTCGTGGTGGGG tggat aactaggg

GGCGGCATCTCAGGTATGGCAGCAGCCAAACTTCTGCATGACTCTGGACTGAATGT

GGTTGTTCTGGAAGCCCGGGACCGTGTGGGAGGCAGGACTTACACTCTTAGGAACC

AAAAGGTTAAATATGTGGACCTTGGAGGATCCTATGTTGGACCAACCCAGAATCGT

ATCTTGAGATTAGCCAAGGAGCTAGGATTGGAGACCTACAAAGTGAATGAGGTTGA

GCGTCTGATCCACCATGTAAAGGGCAAATCATACCCCTTCAGGGGGCCATTCCCAC

CTGTATGGAATCCAATTACCTACTTAGATCATAACAACTTTTGGAGGACAATGGAT

Monoami GACATGGGGCGAGAGATTCCGAGTGATGCCCCATGGAAGGCTCCCCTTGCAGAAG

H227G ne oxidase AGTGGGACAACATGACAATGAAGGAGCTACTGGACAAGCTCTGCTGGACTGAATCT B GCAAAGCAGCTTGCCACTCTCTTTGTGAACCTGTGTGTCACTGCAGAGACCCATGA

GGTCTCTGCTCTCTGGTTCCTGTGGTATGTGAAGCAGTGTGGAGGCACAACAAGAA

TCATCTCGACAACAAATGGAGGACAGGAGAGGAAATTTGTGGGCGGATCTGGTCA

AGTGAGTGAGCGGATAATGGACCTCCTTGGAGACCGAGTGAAGCTGGAGAGGCCT

GTGATCTACATTGACCAGACAAGAGAAAATGTCCTTGTGGAGACCCTAAACCATGA

GATGTATGAGGCTAAATATGTGATTAGTGCTATTCCTCCTACTCTGGGCATGAAGAT

TCACTTCAATCCCCCTCTGCCAATGATGAGAAACCAGATGATCACTCGTGTGCCTTT

GGGTTCAGTCATCAAGTGTATAGTTTATTATAAAGAGCCTTTCTGGAGGAAAAAGG

ATTACTGTGGAACCATGAT

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GGGGGGGGGGGGACCACTTGGCCTGCCTCCGTCCCGCCGCGCCACTTGGCCTGCCT cgcgggatcctctccaccagagaa ggccaagctttcaacttttcgaaga

CCGTCCCGCCGCGCCACTTCGCCTGCCTCCGTCCCCCGCCCGCCGCGCCATGCCTGT gaagggg tcctgagc

GGCCGGCTCGGAGCTGCCGCGCCGGCCCTTGCCCCCCGCCGCACAGGAGCGGGACG

CCGAGCCGCGTCCGCCGCACGGGGAGCTGCAGTACCTGGGGCAGATCCAACACATC

CTCCGCTGCGGCGTCAGGAAGGACGACCGCACGGGCACCGGCACCCTGTCGGTATT

CGGCATGCAGGCGCGCTACAGCCTGAGAGATGAATTCCCTCTGCTGACAACCAAAC

GTGTGTTCTGGAAGGGTGTTTTGGAGGAGTTGCTGTGGTTTATCAAGGGATCCACA

AATGCTAAAGAGCTGTCTTCCAAGGGAGTGAAAATCTGGGATGCCAATGGATCCCG

Thymidyl AGACTTTTTGGACAGCCTGGGATTCTCCACCAGAGAAGAAGGGGACTTGGGCCCAG

H230B ate TTTATGGCTTCCAGTGGAGGCATTTTGGGGCAGAATACAGAGATATGGAATCAGAT synthase TATTCAGGACAGGGAGTTGACCAACTGCAAAGAGTGATTGACACCATCAAAACCA

ACCCTGACGACAGAAGAATCATCATGTGCGCTTGGAATCCAAGAGATCTTCCTCTG

ATGGCGCTGCCTCCATGCCATGCCCTCTGCCAGTTCTATGTGGTGAACAGTGAGCTG

TCCTGCCAGCTGTACCAGAGATCGGGAGACATGGGCCTCGGTGTGCCTTTCAACAT

CGCCAGCTACGCCCTGCTCACGTACATGATTGCGCACATCACGGGCCTGAAGCCAG

GTGACTTTATACACACTTTGGGAGATGCACATATTTACCTGAATCACATCGAGCCAC

TGAAAATTCAGCTTCAGCGAGAACCCAGACCTTTCCCAAAGCTCAGGATTCTTCGA

AAAGTTGAGAAAATTGATGACTTCAAAGCTGAAGACTTTCAGATTGAAGGGTACAA

TCCGCATCCAACTA

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CGCGGAGGCTGCACGGAGAGCGGTGCCCGCGTCAGTGACCGAAGGAAGAGACCAA cgcgggatccagctggctgagcag ggccctgcaggcagtgcaggat

GATGAATACAGAGCCCGAGAGGAAGTTTGGCGTGGTGGTGGTTGGTGTTGGCCGAG aaagga gttte

CCGGCTCCGTGCGGATGAGGGACTTGCGGAATCCACACCCTTCCTCAGCGTTCCTG

AACCTGATTGGCTTCGTGTCGAGAAGGGAGCTCGGGAGCATTGATGGAGTCCAGCA

GATTTCTTTGGAGGATGCTCTTTCCAGCCAAGAGGTGGAGGTCGCCTATATCTGCAG

TGAGAGCTCCAGCCATGAGGACTACATCAGGCAGTTCCTTAATGCTGGCAAGCACG

TCCTTGTGGAATACCCCATGACACTGTCATTGGCGGCCGCTCAGGAACTGTGGGAG

CTGGCTGAGCAGAAAGGAAAAGTCTTGCACGAGGAGCATGTTGAACTCTTGATGGA

GGAATTCGCTTTCCTGAAAAAAGAAGTGGTGGGGAAAGACCTGCTGAAAGGGTCG

Biliverdin

,H234B reductase CTCCTCTTCACAGCTGGCCCGTTGGAAGAAGAGCGGTTTGGCTTCCCTGCATTCAGC

GGCATCTCTCGCCTGACCTGGCTGGTCTCCCTCTTTGGGGAGCTTTCTCTTGTGTCTG

CCACTTTGGAAGAGCGAAAGGAAGATCAGTATATGAAAATGACAGTGTGTCTGGA

GACAGAGAAGAAAAGTCCACTGTCATGGATTGAAGAAAAAGGACCTGGTCTAAAA

CGAAACAGATATTTAAGCTTCCATTTCAAGTCTGGGTCCTTGGAGAATGTGCCAAA

TGTAGGAGTGAATAAGAACATATTTCTGAAAGATCAAAATATATTTGTCCAGAAAC Ui TCTTGGGCCAGTTCTCTGAGAAGGAACTGGCTGCTGAAAAGAAACGCATCCTGCAC

TGCCTGGGGCTTGCAGAAGAAATCCAGAAATATTGCTGTTCAAGGAAGTAAGAGG

AGGAGGTGATGTAGCACTTCCAAGATGGCACCAGCATTTGGTTCTTCTCAAGAGTT

GACCATTATCTCTATTC

ON ON

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GAATTCCGGGAGAAGTGACCAGAGCAATTTCTGCTTTTCACAGGGCGGGTTTCTCA cgcgggatccggcattttggaccaa ggccaagcttgggacagctttccg

ACGGTGACTTGTGGGCAGTGCCTTCTGCTGAGCGAGTCATGGCCCGAAGGCAGAAC gcaga acttc

TAACTGTGCCTGCAGTCTTCACTCTCAGGATGCAGCCGAGGTGGGCCCAAGGGGCC

ACGATGTGGCTTGGAGTCCTGCTGACCCTTCTGCTCTGTTCAAGCCTTGAGGGTCAA

GAAAACTCTTTCACAATCAACAGTGTTGACATGAAGAGCCTGCCGGACTGGACGGT

GCAAAATGGGAAGAACCTGACCCTGCAGTGCTTCGCGGATGTCAGCACCACCTCTC

ACGTCAAGCCTCAGCACCAGATGCTGTTCTATAAGGATGACGTGCTGTTTTACAAC

Platelet/en

ATCTCCTCCATGAAGAGCACAGAGAGTTATTTTATTCCTGAAGTCCGGATCTATGAC dothelial

TCAGGGACATATAAATGTACTGTGATTGTGAACAACAAAGAGAAAACCACTGCAG cell

H261A AGTACCAGCTGTTGGTGGAAGGAGTGCCCAGTCCCAGGGTGACACTGGACAAGAA adhesion

AGAGGCCATCCAAGGTGGGATCGTGAGGGTCAACTGTTCTGTCCCAGAGGAAAAG molecule-

GCCCCAATACACTTCACAATTGAAAAACTTGAACTAAATGAAAAAATGGTCAAGCT

1

GAAAAGAGAGAAGAATTCTCGAGACCAGAATTTTGTGATACTGGAATTCCCCGTTG

AGGAACAGGACCGCGTTTTATCCTTCCGATGTCAAGCTAGGATCATTTCTGGGATCC

ATATGCAGACCTCAGAATCTACCAAGAGTGAACTGGTCACCGTGACGGAATCCTTC

TCTACACCCAAGTTCCACATCAGCCCCACCGGAATGATCATGGAAGGAGCTCAGCT

CCACATTAAGTGCACCATTCAAGTGACTCACCTGGCCCAGGAGTTTCCAGAAATCA

TAATTCAGAAGGACAAGGCGATTGTGGCCCACAACAGACATGGCAACAAGGCTGT

GTACTCAGTCATGGCCA

ON oe

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GGGGAGATTCGGGACCATGGCACCTGTGCACGGCGACGACTCTCTGTCAGATTCAG cgcgggatcctgccctacatgtgga ggccaagctttagccacttcggga

GGAGTTTTGTATCTTCTCGAGCCCGGCGAGAAAAAAAATCAAAGAAGGGGCGCCA actga

AGAAGCCCTAGAAAGACTGAAAAAGGCTAAAGCTGGTGAGAAGTATAAATATGAA

GTCGAGGACTTCACAGGTGTTTATGAAGAAGTTGATGAAGAACAGTATTCGAAGCT

GGTTCAGGCACGCCAGGATGATGACTGGATTGTGGATGATGATGGTATTGGCTATG

TGGAAGATGGCCGAGAGATTTTTGATGATGACCTTGAAGATGATGCCCTTGATGCT

GATGAGAAAGGAAAAGATGGTAAAGCACGCAATAAAGACAAGAGGAATGTAAAG

AAGCTCGCAGTGACAAAACCGAACAACATTAAGTCAATGTTCATTGCTTGTGCTGG

DNA AAAGAAAACTGCAGATAAAGCTGTAGACTTGTCCAAGGATGGTCTGCTAGGTGACA

H27A polymeras TTCTACAGGATCTTAACACTGAGACACCTCAAATAACTCCACCACCTGTAATGATA e alpha CTGAAGAAGAAAAGATCCATTGGAGCTTCACCGAATCCTTTCTCTGTGCACACCGC

CACGGCAGTTCCTTCAGGAAAAATTGCTTCCCCTGTCTCCAGAAAGGAGCCTCCATT

AACTCCTGTTCCTCTTAAACGTGCTGAATTTGCTGGCGATGATGTACAGGTCGAGAG

TACAGAAGAAGAGCAGGAGTCAGGGGCAATGGAGTTTGAAGATGGTGACTTTGAT

GAGCCCATGGAAGTTGAAGAGGTGGACCTGGAGCCTATGGCTGCCAAGGCTTGGG

ACAAAGAGAGTGAGCCAGCAGAGGAAGTGAAACAAGAGGCGGATTCTGGGAAAG

GGACCGTGTCCTACTTAGGAAGTTTTCTCCCGGATGTCTCTTGTTGGGACATTGATC

AAGAAGGTGATAGCAGTTTCTCAGTGCAAGAAGTTCAAGTGGATTCCAGTCACCTC

CCATTGGTAAAAGGGGCAGAT

-4

©

Table 1

NumeriArray Sequence Left PCR primer Right PCR primer cal ref. name sequence sequence

GGCTGAGGCAGCCGCGCAGGTCGCAGGGCCAGCGTCCGCGAGGACGGCCCGGCTG cgcgggatcctctctttgctttcacta ggccctgcagctgatcagctgcc

GGCGGCCCCGAGCTCTGTGGCGCTGTGGAGGAGCGGGAGCGCGGCCGAGGAAGCG tcccagg acctca

GGGCGCCGAGGGGGTCGGCGGCCTTCGGGAAATTTCCGCCGACCCTTCGCTCCCGG

CTCTAAAAGTTCCTGATTTCCTATTTCCTTTTAAATCCCGAGTGGCTGTTAGCTCTTC

GCCTGCACTTTTTCTTCCCCAGGAGATAAGGGGGAGTGTGAGGAACGGAGCGAATA

ATATAAAAAAGGATTTCCTCCCGGAAGAGAGCGGCAGTTCGGAGAGATTTTTCTTA

AGGAAGCAGAAGCGGCGTTTGCGGCCGCTGCAGGCGCCGGGCCCTGCCGGCCACA

CTATGCGCGAGCCGGCCCCGGGCTGCTGAGGCGCGGGGACGCGGAAGCGGAGGCC

Protein-

GAGCGCGCCGGGCTCCCGCGCTCGCGAGCGAGTTTTGTCCGCCCGGCGGCGGTGGC tyrosine

H307F GGGGGGATGGAGCCCGCGACCGCGCCCCGGCCCGACATGGCGCCGGAGCTGACCC phosphata se CGGAGGAGGAGCAGGCTACCAAGCAGTTTCTCGAAGAGATTAACAAGTGGACAGT

TCAGTACAATGTTTCCCCGCTGTCTTGGAATGTGGCTGTCAAGTTCCTCATGGCAAG

GAAGTTTGATGTGCTCCGTGCCATAGAATTGTTCCACTCCTACAGAGAAACTCGAA

GGAAGGAAGGCATTGTAAAGCTGAAACCTCATGAGGAACCTCTTCGTTCTGAGATC

-4

Ul CTCAGTGGAAAATTCACCATCTTAAATGTTCGGGACCCAACAGGAGCCTCCATTGC

CCTCTTTACTGCCAGGTTGCATCATCCCCACAAGTCAGTCCAACATGTGGTACTTCA

GGCTCTGTTTTACTTGCTAGACAGAGCTGTGGATAGCTTTGAAACTCAGAGGAATG

GACTGGTGTTTATCTATGACATGTGTGGTTCTAATTATGCCAACTTTGAGCTGGATC

TTGGCAAGAAAGTCCTA

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CTGCTGGTGGGCAGAGAAGACAGAAACGACATGAGCACAGCAGGAAAAGTAATCA cgcgggatccagttgggtgccact ggccaagcttctgttgctccagatc

AATGCAAAGCAGCTGTGCTATGGGAGGTAAAGAAACCCTTTTCCATTGAGGATGTG gaatgc tgtaggg

GAGGTTGCACCTCCTAAGGCTTATGAAGTTCGCATTAAGATGGTGGCTGTAGGAAT

CTGTCGCACAGATGACCACGTGGTTAGTGGCAACCTGGTGACCCCCCTTCCTGTGAT

TTTAGGCCATGAGGCAGCCGGCATCGTGGAGAGTGTTGGAGAAGGGGTGACTACA

GTCAAACCAGGTGATAAAGTCATCCCGCTCTTTACTCCTCAGTGTGGAAAATGCAG

AGTTTGTAAAAACCCGGAGAGCAACTACTGCTTGAAAAATGATCTAGGCAATCCTC

GGGGGACCCTGCAGGATGGCACCAGGAGGTTCACCTGCAGGGGGAAGCCCATTCA

Alcohol CCACTTCCTTGGCACCAGCACCTTCTCCCAGTACACGGTGGTGGATGAGAATGCAG

H318A dehydroge TGGCCAAAATTGATGCAGCCTCGCCCCTGGAGAAAGTCTGCCTCATTGGCTGTGGA nase 2 TTCTCGACTGGTTATGGGTCTGCAGTTAACGTTGCCAAGGTCACCCCAGGCTCTACC

TGTGCTGTGTTTGGCCTGGGAGGGGTCGGCCTATCTGCTGTTATGGGCTGTAAAGCA

GCTGGAGCAGCCAGAATCATTGCGGTGGACATCAACAAGGACAAAAAAGCAAAGG

CCAAAGAGTTGGGTGCCACTGAATGCATCAACCCTCAAGACTACAAGAAACCCATC

-4 Ul CAGGAAGTGCTAAAGGAAATGACTGATGGAGGTGTGGATTTTTCGTTTGAAGTCAT

CGGTCGGCTTGACACCATGATGGCTTCCCTGTTATGTTGTCATGAGGCATGTGGCAC

AAGCGTCATCGTAGGGGTACCTCCTGCTTCCCAGAACCTCTCAATAAACCCTATGCT

GCTACTGACTGGACGCACCTGGAAGGGGGCTGTTTATGGTGGCTTTAAGAGTAAAG

AAGGTATCCCAAAAC

-4 -4

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CCTGGGTCCTCTCGGCGCCAGAGCCGCTCTCCGCATCCCAGGACAGCGGTGCGGCC cgcgggatccagggtctgcactcc ggccaagcttcctaagctgtcggc

CTCGGCCGGGGCGCCCACTCCGCAGCAGCCAGCGAGCCAGCTGCCCCGTATGACCG caaacc tcagg

CGCCGGGCGCCGCCGGGCGCTGCCCTCCCACGACATGGCTGGGCTCCCTGCTGTTG

TTGGTCTGTCTCCTGGCGAGCAGGAGTATCACCGAGGAGGTGTCGGAGTACTGTAG

CCACATGATTGGGAGTGGACACCTGCAGTCTCTGCAGCGGCTGATTGACAGTCAGA

TGGAGACCTCGTGCCAAATTACATTTGAGTTTGTAGACCAGGAACAGTTGAAAGAT

CCAGTGTGCTACCTTAAGAAGGCATTTCTCCTGGTACAAGACATAATGGAGGACAC

CATGCGCTTCAGAGATAACACCGCCAATCCCATCGCCATTGTGCAGCTGCAGGAAC

Colony- TCTCTTTGAGGCTGAAGAGCTGCTTCACCAAGGATTATGAAGAGCATGACAAGGCC

H324B stimulatin TGCGTCCGAACTTTCTATGAGACACCTCTCCAGTTGCTGGAGAAGGTCAAGAATGT g factor- 1 CTTTAATGAAACAAAGAATCTCCTTGACAAGGACTGGAATATTTTCAGCAAGAACT

GCAACAACAGCTTTGCTGAATGCTCCAGCCAAGATGTGGTGACCAAGCCTGATTGC

AACTGCCTGTACCCCAAAGCCATCCCTAGCAGTGACCCGGCCTCTGTCTCCCCTCAT

CAGCCCCTCGCCCCCTCCATGGCCCCTGTGGCTGGCTTGACCTGGGAGGACTCTGA

-4 oe GGGAACTGAGGGCAGCTCCCTCTTGCCTGGTGAGCAGCCCCTGCACACAGTGGATC

CAGGCAGTGCCAAGCAGCGGCCACCCAGGAGCACCTGCCAGAGCTTTGAGCCGCC

AGAGACCCCAGTTGTCAAGGACAGCACCATCGGTGGCTCACCACAGCCTCGCCCCT

CTGTCGGGGCCTTCAACCCCGGGATGGAGGATATTCTTGACTCTGCAATGGGCACT

AATTGGGTCCCAGAAG

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

TAATTAACTTTTTAAAAGAAGCTAATAAGCATGGATTCCTGGTTCATTCTTGTTCTG cgcgggatccagttaagcggggcj ggccaagctttgctcagggcaca

CTCGGCAGTGGTCTGATATGTGTCAGTGCCAACAATGCTACCACAGTTGCACCTTCT aagaga aaggtc

GTAGGAATTACAAGATTAATTAACTCATCAACGGCAGAACCAGTTAAAGAAGAGG

CCAAAACTTCAAATCCAACTTCTTCACTAACTTCTCTTTCTGTGGCACCAACATTCA

GCCCAAATATAACTCTGGGACCCACCTATTTAACCACTGTCAATTCTTCAGACTCTG

ACAATGGGACCACAAGAACAGCAAGCACCAATTCTATAGGCATTACAATTTCACCA

AATGGAACGTGGCTTCCAGATAACCAGTTCACGGATGCCAGAACAGAACCCTGGG

Protein AGGGGAATTCCAGCACCGCAGCAACCACTCCAGAAACTTTCCCTCCTTCAGATGAG tyrosine ACACCAATTATTGCGGTGATGGTGGCCCTGTCCTCTCTGCTAGTGATCGTGTTTATT

H334B ATCATAGTTTTGTACATGTTAAGGTTTAAGAAATACAAGCAAGCTGGGAGCCATTC phosphata

CAATTCTTTCCGCTTATCCAACGGCCGCACTGAGGATGTGGAGCCCCAGAGTGTGC se alpha

CACTTCTGGCCAGATCCCCAAGCACCAACAGGAAATACCCACCCCTGCCCGTGGAC

AAGCTGGAAGAGGAAATTAACCGGAGAATGGCAGACGACAATAAGCTCTTCAGGG

AGGAATTCAACGCTCTCCCTGCATGTCCTATCCAGGCCACCTGTGAGGCTGCTTCCA oe © AGGAGGAAAACAAGGAAAAAAATCGATATGTAAACATCTTGCCTTATGACCACTCT

AGAGTCCACCTGACACCGGTTGAAGGGGTTCCAGATTCTGATTACATCAATGCTTC

ATTCATCAACGGTTACCAAGAAAAGAACAAATTCATTGCTGCACAAGGACCAAAA

GAAGAAACGGTGAATGATTTCTGGCGGATGATCTGGGAACAAAACACAGCCACCA

TCGTCATGGTTACCA

Table 1

I NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

ATGGGTCTGGAGCTCTACCTGGACCTGCTGTCCCAGCCCTGCCGCGCTGTTTACATC atcgcgggatccacggagagtgtg ttggccaagcttgggtctgcaggt

TTTGCCAAGAAGAACGACATTCCCTTCGAGCTGCGCATCGTGGATCTGATTAAAGG gccatcc ggaagt

TCAGCACTTAAGCGATGCCTTTGCCCAGGTGAACCCCCTCAAGAAGGTGCCGGCCT

TGAAGGACGGGGACTTCACCTTGACGGAGAGTGTGGCCATCCTGCTCTACCTGACG

CGCAAATATAAGGTCCCTGACTACTGGTACCCTCAGGACCTGCAGGCCCGTGCCCG

TGTGGATGAGTACCTGGCATGGCAGCACACGACTCTGCGGAGAAGCTGCCTCCGGG

CCTTGTGGCATAAGGTGATGTTCCCTGTGTTCCTGGGTGGGCCAGTATCTCCCCAGA

Glutathion CACTGGCAGCCACCCTGGCAGAGTTGGATGTGACCCTGCAGTTGCTCGAGGACAAG e S-

H338A TTCCTCCAGAACAAGGCCTTCCTTACTGGTCCTCACATCTCCTTAGCTGACCTCGTA transferas GCCATCACGGAGCTGATGCATCCCGTGGGTGCTGGCTGCCAAGTCTTCGAAGGCCG e theta-1 ACCCAAGCTGGCCACATGGCGGCAGCGCGTGGAGGCAGCAGTGGGGGAGGACCTC

TTCCAGGAGGCCCATGAGGTCATTCTGAAGGCCAAGGACTTCCCACCTGCAGACCC

CACCATAAAGCAGAAGCTGATGCCCTGGGTGCTGGCCATGATCCGGTGAGCTGGGA

AACCTCACCCTTGCACCGTCCTCAGCAGTCCACAAAGCATTTTCATTTCTAATGGCC

CATGGGAGCCAGGCCCAGAAAGCAGGAATGGCTTGCTTAAGACTTGCCCAAGTCCC

AGAGCACCTCACCTCCCGAAGCCACCATCCCCACCCTGTCTTCCACAGCCGCCTGA

AAGCCACAATGAGAATGATGCACACTGAGGCCTTGTGTCCTTTAATCACTGCATTTC

ATTTTGATTTTGGATAATAAACCTGGCTCAGCCTGAGCCTCTGCTTCT

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

AAGTGCTGCGAGCCCTGGGCCACGCTGGCCGTGCTGGCAGTGGGCCGCCTCGATCC cgcgggatccaatttggcgacgtaa ggccaagcttacagcctccgggc

CTCTGCAGTCTTTCCCTTGAGGCTCCAAGACCAGCAGGTGAGGCCTCGCGGCGCTG ttccc tet

AAACCGTGAGGCCCGGACCACAGGCTCCAGATGGACCCTGGGAAGGACAAAGAGG

GGGTGCCCCAGCCCTCAGGGCCGCCAGCAAGGAAGAAATTTGTGATACCCCTCGAC

GAGGATGAGGTCCCTCCTGGAGTGGCCAAGCCCTTATTCCGATCTACACAGAGCCT

TCCCACTGTGGACACCTCGGCCCAGGCGGCCCCTCAGACCTACGCCGAATATGCCA

TCTCACAGCCTCTGGAAGGGGCTGGGGCCACGTGCCCCACAGGGTCAGAGCCCCTG

ERCC 1 GCAGGAGAGACGCCCAACCAGGCCCTGAAACCCGGGGCAAAATCCAACAGCATCA

TTGTGAGCCCTCGGCAGAGGGGCAATCCCGTACTGAAGTTCGTGCGCAACGTGCCC (excision

H33A TGGGAATTTGGCGACGTAATTCCCGACTATGTGCTGGGCCAGAGCACCTGTGCCCT repair

GTTCCTCAGCCTCCGCTACCACAACCTGCACCCAGACTACATCCATGGGCGGCTGC protein)

AGAGCCTGGGGAAGAACTTCGCCTTGCGGGTCCTGCTTGTCCAGGTGGATGTGAAA

GATCCCCAGCAGGCCCTCAAGGAGCTGGCTAAGATGTGTATCCTGGCCGACTGCAC oe ATTGATCCTCGCCTGGAGCCCCGAGGAAGCTGGGCGGTACCTGGAGACCTACAAGG

Kl CCTATGAGCAGAAACCAGCGGACCTCCTGATGGAGAAGCTAGAGCAGGACTTCGTC

TCCCGGGTGACTGAATGTCTGACCACCGTGAAGTCAGTCAACAAAACGGACAGTCA

GACCCTCCTGACCACATTTGGATCTCTGGAACAGCTCATCGCCGCATCAAGAGAAG

ATCTGGCCTTATGCCCAGGCCTGGGCCCTCAGAAAGCCCGGAGGCTGTTTGATGTC

CTGCACGAGCCCTTCTTG

oe

Ul

oe

Ul

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

GCGCCTGTCAGGGAAGCGGCGCGCGCGCGCGGGCGGCGGGCGGGCTGGGGATCCG atcgcgggatccgcgcctttcccaa ttggccaagcttgaaaacagcaa

CCGCGCAGTGCCAGCGCCAGCGCCAGACCCGCGCCCCGCGCTCTCCGGCCCGTCGC ctctagc accaggg

CTGCCTTGGGACTCGCGAGCCCGCACTCCCGCCCTGCCTGTTCGCTGCCCGAGTATG

GAGCTGCTGTGTTGCGAAGGCACCCGGCACGCGCCCCGGGCCGGGCCGGACCCGC

GGCTGCTGGGGGACCAGCGTGTCCTGCAGAGCCTGCTCCGCCTGGAGGAGCGCTAC

GTACCCCGCGCCTCCTACTTCCAGTGCGTGCAGCGGGAGATCAAGCCGCACATGCG

GAAGATGCTGGCTTACTGGATGCTGGAGGTATGTGAGGAGCAGCGCTGTGAGGAG

GAAGTCTTCCCCCTGGCCATGAACTACCTGGATCGCTACCTGTCTTGCGTCCCCACC

CGAAAGGCGCAGTTGCAGCTCCTGGGTGCGGTCTGCATGCTGCTGGCCTCCAAGCT

H348C CyclinD3 GCGCGAGACCACGCCCCTGACCATCGAAAAACTGTGCATCTACACCGACCACGCTG

TCTCTCCCCGCCAGTTGCGGGACTGGGAGGTGCTGGTCCTAGGGAAGCTCAAGTGG

GACCTGGCTGCTGTGATTGCACATGATTTCCTGGCCTTCATTCTGCACCGGCTCTCT

CTGCCCCGTGACCGACAGGCCTTGGTCAAAAAGCATGCCCAGACCTTTTTGGCCCT

CTGTGCTACAGATTATACCTTTGCCATGTACCCGCCATCCATGATCGCCACGGGCAG oe

CATTGGGGCTGCAGTGCAAGGCCTGGGTGCCTGCTCCATGTCCGGGGATGAGCTCA

CAGAGCTGCTGGCAGGGATCACTGGCACTGAAGTGGACTGCCTGCGGGCCTGTCAG

GAGCAGATCGAAGCTGCACTCAGGGAGAGCCTCAGGGAAGCCTCTCAGACCAGCT

CCAGCCCAGCGCCCAAAGCCCCCCGGGGCTCCAGCAGCCAAGGGCCCAGCCAGAC

CAGCACTCCTACAGATG

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CGTTGGCCAAATTGACAAGAGCGAGAGGTATACTGCGTTCCATCCCGACCNGGGGC atcgcgggatcccattctcatcgggt ttggccaagcttcctggaagaaa

CACGGTACTGGGCCCTGTTTCCCCCTCCTCGGCCCCCGAGAGCCAGGGTCCGCCTTC cctcca gtgagcc

TGCAGGGTTCCCAGGCCCCCGCTCCAGGGCCGGGCTGACCCGACTCGCTGGCGCTT

CATGGAGAACTTCCAAAAGGTGGAAAAGATCGGAGAGGGCACGTACGGAGTTGTG

TACAAAGCCAGAAACAAGTTGACGGGAGAGGTGGTGGCGCTTAAGAAAATCCGNN

TGGACACTGAGACTGAGGGTGTGCCCAGTACTGCCATCCGAGAGATCTCTCTGCTT

AAGGAGCTTAACCATCCTAATATTGTCAAGCTGCTGGATGTCATTCACACAGAAAA

TAAACTCTACCTGGTTTTTGAATTTCTGCACCAAGATCTCAAGAAATTCATGGATGC

Cyclin CTCTGCTCTCACTGGCATTCCTCTTCCCCTCATCAAGAGCTATCTGTTCCAGCTGCTC

H349A dependent CAGGGCCTAGCTTTCTGCCATTCTCATCGGGTCCTCCACCGAGACCTTAAACCTCAG kinase 2 AATCTGCTTATTAACACAGAGGGGGCCATCAAGCTAGCAGACTTTGGACTAGCCAG

AGCTTTTGGAGTCCCTGTTCGTACTTACACCCATGAGGTGGTGACCCTGTGGTACCG

AGCTCCTGAAATCCTCCTGGGCTGCAAATATTATTCCACAGCTGTGGACATCTGGA oe GCCTGGGCTGCATCTTTGCTGAGATGGTGACTCGCCGGGCCCTATTCCCTGGAGATT

-4 CTGAGATTGACCAGCTCTTCCGGATCTTTCGGACTCTGGGGACCCCAGATGAGGTG

GTGTGGCCAGGAGTTACTTCTATGCCTGATTACAAGCCAAGTTTCCCCAAGTGGGC

CCGGCAAGATTTTAGTAAAGTTGTACCTCCCCTGGATGAAGATGGACGGAGCTTGT

TATCGCAAATGCTGCACTACGACCCTAACAAGCGGATTTCGGCCAAGGCAGCCCTG

GCTCACCCTTT

oe oe

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

TGTGGAGGTCAGAGTGGAAGCAGGTGTGAGAGGGTCCAGCAGAAGGAAACATGGC atcgcgggatcccattttctcatccc ttggccaagcttgagtcgccctca

TGCCAAAGTGTTTGAGTCCATTGGCAAGTTTGGCCTGGCCTTAGCTGTTGCAGGAG g ggg cagagat

GCGTGGTGAACTCTGCCTTATATAATGTGGATGCTGGGCACAGAGCTGTCATCTTTG

ACCGATTCCGTGGAGTGCAGGACATTGTGGTAGGGGAAGGGACTCATTTTCTCATC

CCGTGGGTACAGAAACCAATTATCTTTGACTGCCGTTCTCGACCACGTAATGTGCCA

GTCATCACTGGTAGCAAAGATTTACAGAATGTCAACATCACACTGCGCATCCTCTTC

CGGCCTGTCGCCAGCCAGCTTCCTCGCATCTTCACCAGCATCGGAGAGGACTATGA

TGAGCGTGTGCTGCCGTCCATCACAACTGAGATCCTCAAGTCAGTGGTGGCTCGCTT

TGATGCTGGAGAACTAATCACCCAGAGAGAGCTGGTCTCCAGGCAGGTGAGCGAC

H359B Prohibitin GACCTTACAGAGCGAGCCGCCACCTTTGGGCTCATCCTGGATGACGTGTCCTTGAC

ACATCTGACCTTCGGGAAGGAGTTCACAGAAGCGGTGGAAGCCAAACAGGTGGCT

CAGCAGGAAGCAGAGAGGGCCAGATTTGTGGTGGAAAAGGCTGAGCAACAGAAAA

AGGCGGCCATCATCTCTGCTGAGGGCGACTCCAAGGCAGCTGAGCTGATTGCCAAC oe TCACTGGCCACTGCAGGGGATGGCCTGATCGAGCTGCGCAAGCTGGAAGCTGCAGA vo GGACATCGCGTACCAGCTCTCACGCTCTCGGAACATCACCTACCTGCCAGCGGGGC

AGTCCGTGCTCCTCCAGCTGCCCCAGTGAGGGCCCACCCTGCCTGCACCTCCGCGG

GCTGACTGGGCCACAGCCCCGATGATTCTTAACACAGCCTTCCTTCTGCTCCCACCC

CAGAAATCACTGTGAAATTTCATGATTGGCTTAAAGTGAAGGAAATAAAGGTAAAA

TCACTTCAGATCTCT

vo ©

VO Ul

VO ON

VO oe

VO VO

Kl

© ©

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

TTCCGGTTTTTCTCAGGGGACGTTGAAATTATTTTTGTAACGGGAGTCGGGAGAGG gcgcggatcccggtcttcatgcaga ggccaagcttttttgcattcgtgttc

ACGGGGCGTGCCCCGCGTGCGCGCGCGTCGTCCTCCCCGGCGCTCCTCCACAGCTC gactga agtaga

GCTGGCTCCCGCCGCGGAAAGGCGTCATGCCGCCCAAAACCCCCCGAAAAACGGC

CGCCACCGCCGCCGCTGCCGCCGCGGAACCCCCGGCACCGCCGCCGCCGCCCCCTC

CTGAGGAGGACCCAGAGCAGGACAGCGGCCCGGAGGACCTGCCTCTCGTCAGGCT

TGAGTTTGAAGAAACAGAAGAACCTGATTTTACTGCATTATGTCAGAAATTAAAGA

TACCAGATCATGTCAGAGAGAGAGCTTGGTTAACTTGGGAGAAAGTTTCATCTGTG

GATGGAGTATTGGGAGGTTATATTCAAAAGAAAAAGGAACTGTGGGGAATCTGTAT

CTTTATTGCACGAGTTGACCTAGATGAGATGTCGTTCACTTTACTGAGCTACAGAAA

H64A Retinoblas

AACATACGAAATCAGTGTCCATAAATTCTTTAACTTACTAAAAGAAATTGATACCA toma

GTACCAAAGTTGATAATGCTATGTCAAGACTGTTGAAGAAGTATGATGTATTGTTT

GCACTCTTCAGCAAATTGGAAAGGACATGTGAACTTATATATTTGACACAACCCAG

CAGTTCGATATCTACTGAAATAAATTCTGCATTGGTGCTAAAAGTTTCTTGGATCAC

Kl

© ATTTTTATTAGCTAAAGGGGAAGTATTACAAATGGAAGATGATCTGGTGATTTCATT

TCAGTTAATGCTATGTGTCCTTGACTATTTTATTAAACTCTCACCTCCCATGTTGCTC

AAAGAACCATATAAAACAGCTGTTATACCCATTAATGGTTCACCTCGAACACCCAG

GCGAGGTCAGAACAGGAGTGCACGGATAGCAAAACAACTAGAAAATGATACAAGA

ATTATTGAAGTTCTCTGTAAAGAACATGAATGTAATATAGATGAGGTGAAAAATGT

TTATTTCAAAAATT

Kl

©

Kl

Kl

©

Ul

Kl

©

Kl

©

Ul

Kl

©

ON

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

TTGCCGATCCGCCGCCCGTCCACACCCGCCGCCAGCTCACCATGGATGATGATATC cgcgggatccgcggctacagcttc ggccaagcttttgctgatccacat

GCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGA accac gctg

CGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCG

TGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAA

GAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAACTGGG

ACGACATGGAGAAAATCTGGCACCACACCTTCTACAATGAGCTGCGTGTGGCTCCC

GAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCGCG

AGAAGATGACCCAGATCATGTTTGAGACCTTCAACACCCCAGCCATGTACGTTGCT

ATCCAGGCTGTGCTATCCCTGTACGCCTCTGGCCGTACCACTGGCATCGTGATGGAC

H75B, Cr Beta-actin TCCGGTGACGGGGTCACCCACACTGTGCCCATCTACGAGGGGTATGCCCTCCCCCA

TGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACTGACTACCTCATGAAGA

TCCTCACCGAGCGCGGCTACAGCTTCACCACCACGGCCGAGCGGGAAATCGTGCGT

GACATTAAGGAGAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAAGAGATGGCCAC

Kl GGCTGCTTCCAGCTCCTCCCTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCA

©

-4 TCACCATTGGCAATGAGCGGTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCC

TGGGCATGGAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGT

GACGTGGACATCCGCAAAGACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCAC

CATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCA

GCACAATGAAGATCAA

Kl

©

VO

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

ATATTGGAGTAGCAAGAGGCTGGGAAGCCATCACTTACCTTGCACTGAGAAAGAA gcgcgaattccaatttcatttctgtttt ggccaagctttcctgcagttgaac

GACAAAGGCCAGTATGCACAGCTTTCCTCCACTGCTGCTGCTGCTGTTCTGGGGTGT ctggc agcta

GGTGTCTCACAGCTTCCCAGCGACTCTAGAAACACAAGAGCAAGATGTGGACTTAG

TCCAGAAATACCTGGAAAAATACTACAACCTGAAGAATGATGGGAGGCAAGTTGA

AAAGCGGAGAAATAGTGGCCCAGTGGTTGAAAAATTGAAGCAAATGCAGGAATTC

TTTGGGCTGAAAGTGACTGGGAAACCAGATGCTGAAACCCTGAAGGTGATGAAGC

AGCCCAGATGTGGAGTGCCTGATGTGGCTCAGTTTGTCCTCACTGAGGGGAACCCT

Type l CGCTGGGAGCAAACACATCTGACCTACAGGATTGAAAATTACACGCCAGATTTGCC

AAGAGCAGATGTGGACCATGCCATTGAGAAAGCCTTCCAACTCTGGAGTAATGTCA interstitial

H86A CACCTCTGACATTCACCAAGGTCTCTGAGGGTCAAGCAGACATCATGATATCTTTTG collagenas

TCAGGGGAGATCATCGGGACAACTCTCCTTTTGATGGACCTGGAGGAAATCTTGCT

CATGCTTTTCAACCAGGCCCAGGTATTGGAGGGGATGCTCATTTTGATGAAGATGA

AAGGTGGACCAACAATTTCAGAGAGTACAACTTACATCGTGTTGCGGCTCATGAAC

Kl TCGGCCATTCTCTTGGACTCTCCCATTCTACTGATATCGGGGCTTTGATGTACCCTA

© GCTACACCTTCAGTGGTGATGTTCAGCTAGCTCAGGATGACATTGATGGCATCCAA

GCCATATATGGACGTTCCCAAAATCCTGTCCAGCCCATCGGCCCACAAACCCCAAA

AGCATGTGACAGTAAGCTAACCTTTGATGCTATAACTACGATTCGGGGAGAAGTGA

TGTTCTTTAAAGACAGATTCTACATGCGCACAAATCCCTTCTACCCGGAAGTTGAGC

TCAATTTCATTTCTGT

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

GTTTTCTACTTTGCCCGCCCACAGATGTAGTTTTCTCTGCGCGTGTGCGTTTTCCCTC cgcgggatcctagagctgggcagt ggccaagcttgccttgcggacctc

CTCCCCCGCCCTCAGGGTCCACGGCCACCATGGCGTATTAGGGGCAGCAGTGCCTG gaagtg ctat

CGGCAGCATTGGCCTTTGCAGCGGCGGCAGCAGCACCAGGCTCTGCAGCGGCAACC

CCCAGCGGCTTAAGCCATGGCGTGAGTACCGGGGCGGGTCGTCCAGCTGTGCTCCT

GGGGCCGGCGCGGGTTTTGGATTGGTGGGGTGCGGCCTGGGGCCAGGGCGGTGCC

GCCAAGGGGGAAGCGATTTAACGAGCGCCCGGGACGCGTGGTCTTTGCTTGGGTGT

CCCCGAGACGCTCGCGTGCCTGGGATCGGGAAAGCGTAGTCGGGTGCCCGGACTGC

Activating TTCCCCAGGAGCCCTACAGCCCTCGGACCCCGAGCCCCGCAAGGTCCCAGGGGTCT

TGGCTGTTGCCCCACGAAACGTGCAGGAACCAAGATGGCGGCGGCAGGGCGGCGG transcripti

H3Ct CGCGGGCGTGAGTCAAGGGCGGGCGGTGGGCGGGGCGCGGCCGCTGGCCGTATTT on factor 4 GGACGTGGGGACGGAGCGCTTTCCTCTTGGCGGCCGGTGGAAGAATCCCCTGGTCT

CCGTGAGCGTCCATTTTGTGGAACCTGAGTTGCAAGCAGGGAGGGGCAAATACAAC

TGCCCTGTTCCCGATTCTCTAGATGGCCGATCTAGAGAAGTCCCGCCTCATAAGTGG

Kl AAGGATGAAATTCTCAGAACAGCTAACCTCTAATGGGAGTTGGCTTCTGATTCTCA Kl TTCAGGCTTCTCACGGCATTCAGCAGCAGCGTTGCTGTAACCGACAAAGACACCTT

CGAATTAAGCACATTCCTCGATTCCAGCAAAGCACCGCAACATGACCGAAATGAGC

TTCCTGAGCAGCGAGGTGTTGGTGGGGGACTTGATGTCCCCCTTCGACCCGTCGGG

TTTGGGGGCTGAAGAAAGCCTAGGTCTCTTAGATGATTACCTGGAGGTGGCCAAGC

ACTTCAAACCTCATGG

Kl

Ul

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

ATGGACCACCTCGGGGCGTCCCTCTGGCCCCAGGTCGGCTCCCTTTGTCTCCTGCTC cgcgggatcccctgcttccctggaa ggccaagcttggctgttctcataag

GCTGGGGCCGCCTGGGCGCCCCCGCCTAACCTCCCGGACCCCAAGTTCGAGAGCAA gtcct ggttgga

AGCGGCCTTGCTGGCGGCCCGGGGGCCCGAAGAGCTTCTGTGCTTCACCGAGCGGT

TGGAGGACTTGGTGTGTTTCTGGGAGGAAGCGGCGAGCGCTGGGGTGGGCCCGGG

CAACTACAGCTTCTCCTACCAGCTCGAGGATGAGCCATGGAAGCTGTGTCGCCTGC

ACCAGGCTCCCACGGCTCGTGGTGCGGTGCGCTTCTGGTGTTCGCTGCCTACAGCCG

ACACGTCGAGCTTCGTGCCCCTAGAGTTGCGCGTCACAGCAGCCTCCGGCGCTCCG

CGATATCACCGTGTCATCCACATCAATGAAGTAGTGCTCCTAGACGCCCCCGTGGG

Erythropo GCTGGTGGCGCGGTTGGCTGACGAGAGCGGCCACGTAGTGTTGCGCTGGCTCCCGC

H198A ietin CGCCTGAGACACCCATGACGTCTCACATCCGCTACGAGGTGGACGTCTCGGCCGGC receptor AACGGCGCAGGGAGCGTACAGAGGGTGGAGATCCTGGAGGGCCGCACCGAGTGTG

TGCTGAGCAACCTGCGGGGCCGGACGCGCTACACCTTCGCCGTCCGCGCGCGTATG

GCTGAGCCGAGCTTCGGCGGCTTCTGGAGCGCCTGGTCGGAGCCTGTGTCGCTGCT

GACGCCTAGCGACCTGGACCCCCTCATCCTGACGCTCTCCCTCATCCTCGTGGTCAT

CCTGGTGCTGCTGACCGTGCTCGCGCTGCTCTCCCACCGCCGGGCTCTGAAGCAGA

AGATCTGGCCTGGCATCCCGAGCCCAGAGAGCGAGTTTGAAGGCCTCTTCACCACC

CACAAGGGTAACTTCCAGCTGTGGCTGTACCAGAATGATGGCTGCCTGTGGTGGAG

CCCCTGCACCCCCTTCACGGAGGACCCACCTGCTTCCCTGGAAGTCCTCTCAGAGCG

CTGCTGGGGGACGA

Kl Ul

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GAGTTGCCACAGCTCTTCTACTCCACTGCTGTCTATCTTGCCTGCCGGCACCCAGCC atcgcgggatccagccgcacacca ttggccaagctttaggtgctaccct

ACCATGTGGGAGCTCGTGGCTCTCTTGCTGCTTACCCTAGCTTATTTGTTTTGGCCC actatcagt agcctgg

AAGAGAAGGTGCCCTGGTGCCAAGTACCCCAAGAGCCTCCTGTCCCTGCCCCTGGT

GGGCAGCCTGCCATTCCTCCCCAGACATGGCCATATGCATAACAACTTCTTCAAGCT

GCAGAAAAAATATGGCCCCATCTATTCTGTTCGTATGGGCACCAAGACTACAGTGA

TTGTCGGCCACCACCAGCTGGCCAAGGAGGTGCTTATTAAGAAGGGCAAGGACTTC

TCTGGGCGGCCTCAAATGGCAACTCTAGACATCGCGTCCAACAACCGTAAGGGTAT

CGCCTTCGCTGACTCTGGCGCACACTGGCAGCTGCATCGAAGGCTGGCGATGGCCA

Cytochro CCTTTGCCCTGTTCAAGGATGGCGATCAGAAGCTGGAGAAGATCATTTGTCAGGAA

H420B me P450 ATCAGTACATTGTGTGATATGCTGGCCACCCACAACGGACAGTCCATAGACATCTC 17A CTTTCCTGTCTTCGTGGCGGTAACCAATGTCATCTCCTTGATCTGCTTCAATACCTCC

TACAAGAATGGGGACCCTGAGTTGAATGTCATACAGAATTACAATGAAGGCATCAT

AGACAACCTGAGCAAAGACAGCCTGGTGGACCTAGTCCCCTGGTTGAAGATTTTCC

Kl CCAACAAAACCCTGGAAAAATTAAAGAGCCATGTTAAAATACGAAATGATCTGCTG

AATAAAATACTTGAAAATTACAAGGAGAAATTCCGGAGTGACTCTATCACCAACAT

GCTGGACACACTGATGCAAGCCAAGATGAACTCAGATAATGGCAATGCTGGCCCA

GATCAAGATTCAGAGCTGCTTTCAGATAACCACATTCTCACCACCATAGGGGACAT

CTTTGGGGCTGGCGTGGAGACCACCACCTCTGTGGTTAAATGGACCCTGGCCTTCCT

GCTGCACAATC

Kl -4

Kl oe

Kl

VO

Kl Kl

©

Kl Kl

Kl Kl Kl

K K

Ul

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GGAGAACCGTTCGCGGAGGAAAGGCGAACTAGTGTTGGGATGGCCACCAACTGGG cgcgggatccccagctggctggga ggccaagcttctgccacaccacca

GGAGCCTCTTGCAGGATAAACAGCAGCTAGAGGAGCTGGCACGGCAGGCCGTGGA ctaaga ctgc

CCGGGCCCTGGCTGAGGGAGTATTGCTGAGGACCTCACAGGAGCCCACTTCCTCGG

AGGTGGTGAGCTATGCCCCATTCACGCTCTTCCCCTCACTGGTCCCCAGTGCCCTGC

TGGAGCAAGCCTATGCTGTGCAGATGGACTTCAACCTGCTAGTGGATGCTGTCAGC

CAGAACGCTGCCTTCCTGGAGCAAACTCTTTCCAGCACCATCAAACAGGATGACTT

TACCGCTCGTCTCTTTGACATCCACAAGCAAGTCCTAAAAGAGGGCATTGCCCAGA

CTGTGTTCCTGGGCCTGAATCGCTCAGACTACATGTTCCAGCGCAGCGCAGATGGC

Glutathion TCCCCAGCCCTGAAACAGATCGAAATCAACACCATCTCTGCCAGCTTTGGGGGCCT

PHI 1 IB e GGCCTCCCGGACCCCAGCTGTGCACCGACATGTTCTCAGTGTCCTGAGTAAGACCA synthetase AAGAAGCTGGCAAGATCCTCTCTAATAATCCCAGCAAGGGACTGGCCCTGGGAATT

GCCAAAGCCTGGGAGCTCTACGGCTCACCCAATGCTCTGGTGCTACTGATTGCTCA

AGAGAAGGAAAGAAACATATTTGACCAGCGTGCCATAGAGAATGAGCTACTGGCC

Kl AGGAACATCCATGTGATCCGACGAACATTTGAAGATATCTCTGAAAAGGGGTCTCT Kl GGACCAAGACCGAAGGCTGTTTGTGGATGGCCAGGAAATTGCTGTGGTTTACTTCC

GGGATGGCTACATGCCTCGTCAGTACAGTCTACAGAATTGGGAAGCACGTCTACTG

CTGGAGAGGTCACATGCTGCCAAGTGCCCAGACATTGCCACCCAGCTGGCTGGGAC

TAAGAAGGTGCAGCAGGAGCTAAGCAGGCCGGGCATGCTGGAGATGTTGCTCCCT

GGCCAGCCTGAGGCTGTGG

Kl Kl Ul

Kl Kl

ON

Kl Kl -4

Kl Kl oe

Kl Kl

VO

Kl

Ul

©

Kl

Ul

Kl

Ul Kl

Kl

Ul Ul

Kl

Ul

Kl

Ul Ul

Kl

Ul

ON

Kl

Ul -4

Kl

Ul oe

Kl

Ul

VO

Kl

4-

©

Kl

4- Kl

Kl

4- Ui

Kl

4- 4-

Kl

4- Ul

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

CCTGACGCAGTGTCTTGGGCGCTAACGGCGGCGGCGGCCTTGTGTTTAGACTCCAGAACTCCCCACTTGCC cgcgggatcccagacagtggccac ggccaagcttaggccttctcca

GCGTTCTCGCCGCCGCAGGCTCCCGGGACGATGGTGCCCCGCCTGCTGCTQCGCGCCTGGCCCCGGGGCC

CCGCGGTTGGTCCGGGAGCCCCCAGTCGGCCCCTCAGCGCCGGCTCCGGGCCCGGCCAGTACCTGCAGCG ctacga attgg

CAGCATCGTGCCCACCATGCACTACCAGGACAGCCTGCCCAGGCTGCCTATTCCCAAACTTGAAGACACC

ATTAGGAGATACCTCAGTGCACAGAAGCCTCTCTTGAATGATGGCCAGTTCAGGAAAACAGAACAATTTT

GCAAGAGTTTTGAAAATGGGATTGGAAAAGAACTGCATGAGCAGCTGGTTGCTCTGGACAAACAGAATA

AACATACAAGCTACATTTCGGGACCCTGGTTTGATATGTACCTATCTGCTCGAGACTCCGTTGTTCTGAAC

TTTAATCCATTTATGGCTTTCAATCCTGACCCAAAATCTGAGTATAATGACCAGCTCACCCGGGCAACCAA

CATGACTGTTTCTGCCATCCGGTTTCTGAAGACACTCCGGGCTGGCCTTCTGGAGCCAGAAGTGTTCCACT

TGAACCCTGCAAAAAGTGACACTATCACCTTCAAGAGACTCATACGCTTTGTGCCTTCCTCTCTGTCCTGG

TATGGGGCCTACCTGGTCAATGCGTATCCCCTGGATATGTCCCAGTATTTTCGGCTTTTCAACTCAACTCGT

TTACCCAAACCCAGTCGGGATGAACTCπCACTGATGACAAGGCCAGACACCTCCTGGTCCTAAGGAAAG

GAAATTTTTATATCTTTGATGTCCTGGATCAAGATGGGAACATTGTGAGCCCCTCGGAAATCCAGGCACAT

CTGAAGTACATTCTCTCAGACAGCAGCCCCGCCCCCGAGTTTCCCCTGGCATACCTGACCAGTGAGAACC

Carnitine GAGACATCTGGGCAGAGCTCAGGCAGAAGCTGATGAGTAGTGGCAATGAGGAGAGCCTGAGGAAAGTGG palmitoyl- ACTCGGCAGTGTTCTGTCTCTGCCTAGATGACTTCCCCATTAAGGACCTTGTCCACTTGTCCCACAATATGC

Kl TGCATGGGGATGGCACAAACCGCTGGTTTGATAAATCCTTTAACCTCATTATCGCCAAGGATGGCTCTACT

4- H101G CoA GCCGTCCACTTTGAGCACTCTTGGGGTGATGGTGTGGCAGTGCTCAGATTTTTTAATGAAGTATTTAAAGA transferas CAGCACTCAGACCCCTGCCGTCACTCCACAGAGCCAGCCAGCTACCACTGACTCTACTGTCACGGTGCAG

AAACTCAACTrCGAGCTGACTGATGCCTTAAAGACTGGCATCACAGCTGCTAAGGAAAAGTTTGATGCCA

CCATGAAAACCCTCACTATTGACTGCGTCCAGTTTCAGAGAGGAGGCAAAGAATTCCTGAAGAAGCAAAA

GCTGAGCCCTGACGCAGTTGCCCAGCTGGCATTCCAGATGGCCTTCCTGCGGCAGTACGGGCAGACAGTG

GCCACCTACGAGTCCTGTAGCACTGCCGCATTCAAGCACGGCCGCACTGAGACCATCCGCCCGGCCTCCG

TCTATACAAAGAGGTGCTCTGAGGCCTTTGTCAGGGAGCCCTCCAGGCACAGTGCTGGTGAGCTTCAGCA

GATGATGGTTGAGTGCTCCAAGTACCATGGCCAGCTGACCAAAGAAGCAGCAATGGGCCAGGGCTTTGAC

CGACACTTGTTTGCTCTGCGGCATCTGGCAGCAGCCAAAGGGATCATCTTGCCTGAGCTCTACCTGGACCC

TGCATACGGGCAGATAAACCACAATGTCCTGTCCACGAGCACACTGAGCAGCCCAGCAGTGAACCTTGGG

GGCTTTGCCCCTGTGGTCTCTGATGGCTTTGGTGTTGGGTATGCTGTTCATGACAACTGGATAGGCTGCAA

TGTCTCTTCCTACCCAGGCCGCAATGCCCGGGAGTTTCTCCAATGTGTGGAGAAGGCCTTAGAAGACATGT

TTGATGCCTTAGAAGGCAAATCCATCAAAAGTTAACTTCTGGGCAGATGAAAAGCTACCATCACTTCCTC

ATCATGAAAACTGGGAGGCCGGGCATGGTGCTCATGCCTGTAATCCCAGCATTTTGAGAGGCTGAGGCGG

GTGGATCACTTGAGGTCAGGAGTTTGAGACCAACCTGGCCAACATGGTGAAACCTTGTCTCTACTAAAAA

TG

Kl 4- -4

Kl

4- oe

Kl

4-

Kl Ul

©

Kl Ul

Kl Ul Kl

Kl Ul

Ul

Kl Ul

Kl Ul Ul

Kl Ul

ON

Kl Ul -4

Kl Ul oe

Kl Ul

Kl

ON ©

Kl

Kl

ON Kl

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CGACCAAAGCGCCTGAGGACCGGCAACATGGTGCGGTCGGGGAATAAGGCAGCTG cgcgggatccttccaagacaaccat ggccaagcttagctgctgtgtctcc

TTGTGCTGTGTATGGACGTGGGCTTTACCATGAGTAACTCCATTCCTGGTATAGAAT gaagatg cttg

CCCCATTTGAACAAGCAAAGAAGGTGATAACCATGTTTGTACAGCGACAGGTGTTT

GCTGAGAACAAGGATGAGATTGCTTTAGTCCTGTTTGGTACAGATGGCACTGACAA

TCCCCTTTCTGGTGGGGATCAGTATCAGAACATCACAGTGCACAGACATCTGATGC

TACCAGATTTTGATTTGCTGGAGGACATTGAAAGCAAAATCCAACCAGGTTCTCAA

CAGGCTGACTTCCTGGATGCACTAATCGTGAGCATGGATGTGATTCAACATGAAAC

ATP- AATAGGAAAGAAGTTTGAGAAGAGGCATATTGAAATATTCACTGACCTCAGCAGCC dependent GATTCAGCAAAAGTCAGCTGGATATTATAATTCATAGCTTGAAGAAATGTGACATC

H5Dt TCCCTGCAATTCTTCTTGCCTTTCTCACTTGGCAAGGAAGATGGAAGTGGGGACAG helicase II (Ku80) AGGAGATGGCCCCTTTCGCTTAGGTGGCCATGGGCCTTCCTTTCCACTAAAAGGAA

TTACCGAACAGCAAAAAGAAGGTCTTGAGATAGTGAAAATGGTGATGATATCTTTA

GAAGGTGAAGATGGGTTGGATGAAATTTATTCATTCAGTGAGAGTCTGAGAAAACT

Kl GTGCGTCTTCAAGAAAATTGAGAGGCATTCCATTCACTGGCCCTGCCGACTGACCA

TTGGCTCCAATTTGTCTATAAGGATTGCAGCCTATAAATCGATTCTACAGGAGAGA

GTTAAAAAGACTTGGACAGTTGTGGATGCAAAAACCCTAAAAAAAGAAGATATAC

AAAAAGAAACAGTTTATTGCTTAAATGATGATGATGAAACTGAAGTTTTAAAAGAG

GATATTATTCAAGGGTTCCGCTATGGAAGTGATATAGTTCCTTTCTCTAAAGTGGAT

GAGGAACAAATGAAAT

Kl

ON

Kl

ON Ul

Table 1

NumeriArray Left PCR'primer Right PCR primer

Sequence cal ref. name sequence sequence cgcgggatcccgaacataacagag ggccaagctttcaggcatcatcga

GAGTAGCGAAAAGATCTGCTCGAGGCCTGGGTGCTTTGGTGTCGGAGATCCGAGAG tactgcagag tttct

TCGGAGATCGGAGAGTCGGACACAGGACAGTCGGACACCGGACAGTCAAACACCG

GAGAGTTAGACTGGGCTTCTCGGTGGGGACAGGCTCTGGGATAACTACTGTTACAG

CTTTGAAGGGTCAAGGGTGTGCGCTTTTTCTTTCATCCTTCCCTTTCCTGCTGCAGGC

GAGGCCGGTCTGATGCGGATCACTTCCTTTCGCCCACACATTGGCGGAGGAGAAAC

CGGAAAGTTAATCACTGCCCTGCTCTGAGAACTCGGGCCTTTAGGGGCACGTTCGC

CTGCTGACCGGTCTTCTGATCTCCCCATTCTTTTCCATGCAGGAGGATTGGCCACCA

DNA AAGCCTGTTTATTAGCAGCTGCCATTTGTTAAAGAAATTTGGATTATTTTAGAAACA

H23A dependent ATTTGGAAAGAAAAAGAATGGCGTCCGTTTCAGCTCTAACTGAGGAACTGGATTCT helicase ATAACCAGTGAGCTACATGCAGTAGAAATTCAAATTCAAGAACTTACGGAAAGGC

AACAAGAGCTTATTCAGAAAAAAAAAGTCCTGACAAAGAAAATAAAGCAGTGTTT

AGAGGATTCTGATGCCGGGGCAAGCAATGAATATGATTCTTCACCTGCCGCTTGGA

Kl ATAAAGAAGATTTTCCATGGTCTGGTAAAGTTAAAGATATTCTGCAAAATGTCTTTA

AACTGGAAAAGTTCAGACCACTTCAGCTTGAAACTATTAACGTAACAATGGCTGGA

AAGGAGGTATTTCTTGTTATGCCTACAGGAGGTGGAAAGAGCTTATGTTACCAGTT

ACCAGCATTATGTTCAGATGGTTTTACACTCGTCATTTGCCCATTGATCTCTCTTATG

GAAGACCAATTAATGGTTTTAAAACAATTAGGAATTTCAGCAACCATGTTAAATGC

TTCTAGTTC

Kl

ON -4

Kl

ON oe

Kl

ON VO

Kl -4

©

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CCTACTCTATTCAGATATTCTCCAGATTCCTAAAGATTAGAGATCATTTCTCATTCTC gcgcggatccgcattgctgagaac ggccaagcttcagcctggacactg

CTAGGAGTACTCACTTCAGGAAGCAACCAGATAAAAGAGAGGTGCAACGGAAGCC attgcc accatt

AGAACATTCCTCCTGGAAATTCAACCTGTTTCGCAGTTTCTCGAGGAATCAGCATTC

AGTCAATCCGGGCCGGGAGCAGTCATCTGTGGTGAGGCTGATTGGCTGGGCAGGAA

CAGCGCCGGGGCGTGGGCTGAGCACAGCGCTTCGCTCTCTTTGCCACAGGAAGCCT

GAGCTCATTCGAGTAGCGGCTCTTCCAAGCTCAAAGAAGCAGAGGCCGCTGTTCGT

TTCCTTTAGGTCTTTCCACTAAAGTCGGAGTATCTTCTTCCAAGATTTCACGTCTTGG

TGGCCGTTCCAAGGAGCGCGAGGTCGGGATGGATCTTGAAGGGGACCGCAATGGA iMultidrag GGAGCAAAGAAGAAGAACTTTTTTAAACTGAACAATAAAAGTGAAAAAGATAAGA

H53D resistant AGGAAAAGAAACCAACTGTCAGTGTATTTTCAATGTTTCGCTATTCAAATTGGCTTG protein- 1 ACAAGTTGTATATGGTGGTGGGAACTTTGGCTGCCATCATCCATGGGGCTGGACTT

CCTCTCATGATGCTGGTGTTTGGAGAAATGACAGATATCTTTGCAAATGCAGGAAA

TTTAGAAGATCTGATGTCAAACATCACTAATAGAAGTGATATCAATGATACAGGGT

Kl TCTTCATGAATCTGGAGGAAGACATGACCAGGTATGCCTATTATTACAGTGGAATT -4 GGTGCTGGGGTGCTGGTTGCTGCTTACATTCAGGTTTCATTTTGGTGCCTGGCAGCT

GGAAGACAAATACACAAAATTAGAAAACAGTTTTTTCATGCTATAATGCGACAGGA

GATAGGCTGGTTTGATGTGCACGATGTTGGGGAGCTTAACACCCGACTTACAGATG

ATGTCTCTAAGATTAATGAAGTTATTGGTGACAAAATTGGAATGTTCTTTCAGTCAA

TGGCAACATTT

Kl -4 Kl

Kl -4

Ui

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GGGCACGAGCCCTGCCATGTCTCGCCGGAAGCCTGCGTCGGGCGGCCTCGCTGCCT cgcggaattctcagaaaagcaacat ggccaagctttgaactcctctgtcc

CCAGCTCAGCCCCTGCGAGGCAAGCGGTTTTGAGCCGATTCTTCCAGTCTACGGGA cacagtcc cttctgc

AGCCTGAAATCCACCTCCTCCTCCACAGGTGCAGCCGACCAGGTGGACCCTGGCGC

TGCAGCGGCCGCAGCGCCCCCAGCGCCCGCCTTCCCGCCCCAGCTGCCGCCGCACG

TAGCTACAGAAATTGACAGAAGAAAGAAGAGACCATTGGAAAATGATGGGCCTGT

TAAAAAGAAAGTAAAGAAAGTCCAACAAAAGGAAGGAGGAAGTGATCTGGGAAT

GTCTGGCAACTCTGAGCCAAAGAAATGTCTGAGGACCAGGAATGTTTCAAAGTCTC

Mismatch TGGAAAAATTGAAAGAATTCTGCTGCGATTCTGCCCTTCCTCAAAGTAGAGTCCAG repair/bin ACAGAATCTCTGCAGGAGAGATTTGCAGTTCTGCCAAAATGTACTGATTTTGATGA

H91Dr ding TATCAGTCTTCTACACGCAAAGAATGCAGTTTCTTCTGAAGATTCGAAACGTCAAAT protein TAATCAAAAGGACACAACACTTTTTGATCTCAGTCAGTTTGGATCATCAAATACAA

(hMSH3) GTCATGAAAATTTACAGAAAACTGCTTCCAAATCAGCTAACAAACGGTCCAAAAGC

ATCTATACGCCGCTAGAATTACAATACATAGAAATGAAGCAGCAGCACAAAGATG

Kl -4 CAGTTTTGTGTGTGGAATGTGGATATAAGTATAGATTCTTTGGGGAAGATGCAGAG

ATTGCAGCCCGAGAGCTCAATATTTATTGCCATTTAGATCACAACTTTATGACAGCA

AGTATACCTACTCACAGACTGTTTGTTCATGTACGCCGCCTGGTGGCAAAAGGATA

TAAGGTGGGAGTTGTGAAGCAAACTGAAACTGCAGCATTAAAGGCCATTGGAGAC

AACAGAAGTTCACTCTTTTCCCGGAAATTGACTGCCCTTTATACAAAATCTACACTT

ATTGGAGAAGATGTGAAT

Kl -4 Ul

Kl -4

ON

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GAGCGCCGGCGCGGGACCGAGCTGGCGGCGGGCGGCGCGCGCTTCCGAGGCTTCC cgcgtctagatggggattcaaaccg ggccaagcttagggtgaatggcg

TGCTGCTTCTGCCCGAGCCCGCGGCCTCACGCGCGCCCTCTCCCGTGCCATGGCCTG atgac ctgtgt

CAGGCAGGAGCCGCAGCCGCAGGGCCCGCCGCCCGCTGCTGGCGCCGTGGCCTCCT

ATGACTACCTGGTGATCGGGGGCGGCTCGGGCGGGCTGGCCAGCGCGCGCAGGGC

GGCCGAGCTGGGTGCCAGGGCCGCCGTGGTGGAGAGCCACAAGCTGGGTGGCACT

TGCGTGAATGTTGGATGTGTACCCAAAAAGGTAATGTGGAACACAGCTGTCCACTC

TGAATTCATGCATGATCATGCTGATTATGGCTTTCCAAGTTGTGAGGGTAAATTCAA

TTGGCGTGTTATTAAGGAAAAGCGGGATGCCTATGTGAGCCGCCTGAATGCCATCT

Glutathion ATCAAAACAATCTCACCAAGTCCCATATAGAAATCATCCGTGGCCATGCAGCCTTC

H112A e ACGAGTGATCCCAAGCCCACAATAGAGGTCAGTGGGAAAAAGTACACCGCCCCAC reductase ACATCCTGATCGCCACAGGTGGTATGCCCTCCACCCCTCATGAGAGCCAGATCCCC

CCGCAGCGTCATTGTTGGTGCAGGTTACATTGCTGTGGAGATGGCAGGGATCCTGT

Kl CAGCCCTGGGTTCTAAGACATCACTGATGATACGGCATGATAAGGTACTTAGAAGT -4 -4 TTTGATTCAATGATCAGCACCAACTGCACGGAGGAGCTGGAGAACGCTGGCGTGGA

GGTGCTGAAGTTCTCCCAGGTCAAGGAGGTTAAAAAGACTTTGTCGGGCTTGGAAG

TCAGCATGGTTACTGCAGTTCCCGGTAGGCTACCAGTCATGACCATGATTCCAGAT

GTTGACTGCCTGCTCTGGGCCATTGGGCGGGTCCCGAATACCAAGGACCTGAGTTT

AAACAAACTGGGGATTCA

Kl -4 oe

Kl -4 O

Kl oe ©

Kl oe

Kl oe

Kl

Kl oe

Ul

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GTTCCTCTATCTCGTCTTGTTGCTGATTAAAGGTGCCCCTGTCTCCAGTTTTTCTCCA cgcgggatccctcctagcaggcag ggccaagcttaggaacccaagcg

TCTCCTGGGACGTAGCAGGAAATCAGCATCATGGTTGGGTTCAAGGCCACAGATGT caeca agaaag

GCCCCCTACTGCCACTGTGAAGTTTCTTGGGGCTGGCACAGCTGCCTGCATCGCAG

ATCTCATCACCTTTCCTCTGGATACTGCTAAAGTCCGGTTACAGATCCAAGGAGAA

AGTCAGGGGCCAGTGCGCGCTACAGCCAGCGCCCAGTACCGCGGTGTGATGGGCA

CCATTCTGACCATGGTGCGTACTGAGGGCCCCCGAAGCCTCTACAATGGGCTGGTT

GCCGGCCTGCAGCGCCAAATGAGCTTTGCCTCTGTCCGCATCGGCCTGTATGATTCT

GTCAAACAGTTCTACACCAAGGGCTCTGAGCATGCCAGCATTGGGAGCCGCCTCCT

Uncouplin AGCAGGCAGCACCACAGGTGCCCTGGCTGTGGCTGTGGCCCAGCCCACGGATGTGG

H170A g protein TAAAGGTCCGATTCCAAGCTCAGGCCCGGGCTGGAGGTGGTCGGAGATACCAAAG 2 CACCGTCAATGCCTACAAGACCATTGCCCGAGAGGAAGGGTTCCGGGGCCTCTGGA

AAGGGACCTCTCCCAATGTTGCTCGTAATGCCATTGTCAACTGTGCTGAGCTGGTGA

CCTATGACCTCATCAAGGATGCCCTCCTGAAAGCCAACCTCATGACAGATGACCTC

Kl CCTTGCCACTTCACTTCTGCCTTTGGGGCAGGCTTCTGCACCACTGTCATCGCCTCC oe CCTGTAGACGTGGTCAAGACGAGATACATGAACTCTGCCCTGGGCCAGTACAGTAG

CGCTGGCCACTGTGCCCTTACCATGCTCCAGAAGGAGGGGCCCCGAGCCTTCTACA

AAGGGTTCATGCCCTCCTTTCTCCGCTTGGGTTCCTGGAACGTGGTGATGTTCGTCA

CCTATGAGCAGCTGAAACGAGCCCTCATGGCTGCCTGCACTTCCCGAGAGGCTCCC

TTCTGAGCCTCT

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GCTCGAGCCAGCGGCGCCCGGAGAGATTCGGAGATGCAGGCGGCTCGGATGGCCG cgcggaattcctgtatggacaaagg ggccaagcttggccttggagatgc

CGAGCTTGGGGCGGCAGCTGCTGAGGCTCGGGGGCGGAAGCTCGCGGCTCACGGC aaaggagc tttga

GCTCCTGGGGCAGCCCCGGCCCGGCCCTGCCCGGCGGCCCTATGCCGGGGGTGCCG

CTCAGCTGGCTCTGGACAAGTCAGATTCCCACCCCTCTGACGCTCTGACCAGGAAA

AAACCGGCCAAGGCGGAATCTAAGTCCTTTGCTGTGGGAATGTTCAAAGGCCAGCT

CACCACAGATCAGGTGTTCCCATACCCGTCCGTGCTCAACGAAGAGCAGACACAGT

TTCTTAAAGAGCTGGTGGAGCCTGTGTCCCGTTTCTTCGAGGAAGTGAACGATCCC

Very GCCAAGAATGACGCTCTGGAGATGGTGGAGGAGACCACTTGGCAGGGCCTCAAGG long-chain AGCTGGGGGCCTTTGGTCTGCAAGTGCCCAGTGAGCTGGGTGGTGTGGGCCTTTGC

H171D acyl-CoA AACACCCAGTACGCCCGTTTGGTGGAGATCGTGGGCATGCATGACCTTGGCGTGGG dehydroge CATTACCCTGGGGGCCCATCAGAGCATCGGTTTCAAAGGCATCCTGCTCTTTGGCAC nase AAAGGCCCAGAAAGAAAAATACCTCCCCAAGCTGGCATCTGGGGAGACTGTGGCC

GCTTTCTGTCTAACCGAGCCCTCAAGCGGGTCAGATGCAGCCTCCATCCGAACCTCT

Kl GCTGTGCCCAGCCCCTGTGGAAAATACTATACCCTCAATGGAAGCAAGCTTTGGAT oe

Ul CAGTAATGGGGGCCTAGCAGACATCTTCACGGTCTTTGCCAAGACACCAGTTACAG

ATCCAGCCACAGGAGCCGTGAAGGAGAAGATCACAGCTTTTGTGGTGGAGAGGGG

CTTCGGGGGCATTACCCATGGGCCCCCTGAGAAGAAGATGGGCATCAAGGCTTCAA

ACACAGCAGAGGTGTTCTTTGATGGAGTACGGGTGCCATCGGAGAACGTGCTGGGT

GAGGTTGGGAGTGGCTTCA

Kl oe

ON

Kl oe

-4

Kl oe oe

Kl oe vo

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

CTGGGACAGTGAATCGACAATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGC cgcgggatcccaaggacaccgag ggccaagcttttgaacttgacctcg

AGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTGAGGATCCCCAGGGAGATGCTG gaagagg ;g

CCCAGAAGACAGATACATCCCACCATGATCAGGATCACCCAACCTTCAACAAGATC

ACCCCCAACCTGGCTGAGTTCGCCTTCAGCCTATACCGCCAGCTGGCACACCAGTC

CAACAGCACCAATATCTTCTTCTCCCCAGTGAGCATCGCTACAGCCTTTGCAATGCT

CTCCCTGGGGACCAAGGCTGACACTCACGATGAAATCCTGGAGGGCCTGAATTTCA

ACCTCACGGAGATTCCGGAGGCTCAGATCCATGAAGGCTTCCAGGAACTCCTCCGT

ACCCTCAACCAGCCAGACAGCCAGCTCCAGCTGACCACCGGCAATGGCCTGTTCCT

H194B, Alpha 1- CAGCGAGGGCCTGAAGCTAGTGGATAAGTTTTTGGAGGATGTTAAAAAGTTGTACC A antitrypsin ACTCAGAAGCCTTCACTGTCAACTTCGGGGACACCGAAGAGGCCAAGAAACAGAT

CAACGATTACGTGGAGAAGGGTACTCAAGGGAAAATTGTGGATTTGGTCAAGGAG

CTTGACAGAGACACAGTTTTTGCTCTGGTGAATTACATCTTCTTTAAAGGCAAATGG

GAGAGACCCTTTGAAGTCAAGGACACCGAGGAAGAGGACTTCCACGTGGACCAGG

Kl TGACCACCGTGAAGGTGCCTATGATGAAGCGTTTAGGCATGTTTAACATCCAGCAC

© TGTAAGAAGCTGTCCAGCTGGGTGCTGCTGATGAAATACCTGGGCAATGCCACCGC

CATCTTCTTCCTGCCTGATGAGGGGAAACTACAGCACCTGGAAAATGAACTCACCC

ACGATATCATCACCAAGTTCCTGGAAAATGAAGACAGAAGGTCTGCCAGCTTACAT

TTACCCAAACTGTCCATTACTGGAACCTATGATCTGAAGAGCGTCCTGGGTCAACT

GGGCATCACTAAGGTC

Kl

VO Kl

Kl

VO Ul

Kl

VO

Kl

VO Ul

Kl

VO ON

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CTCCAAAACCCTCGTCGACATGGACATGGCCGACTACAGTGCTGCACTGGACCCAG cgcgggatccctttgacccagatgc ggccaagcttttgacgattgtggcg

CCTACACCACCCTGGAATTTGAGAATGTGCAGGTGTTGACGATGGGCAATGACACG caagg acg

TCCCCATCAGAAGGCACCAACCTCAACGCGCCCAACAGCCTGGGTGTCAGCGCCCT

GTGTGCCATCTGCGGGGACCGGGCCACGGGCAAACACTACGGTGCCTCGAGCTGTG

ACGGCTGCAAGGGCTTCTTCCGGAGGAGCGTGCGGAAGAACCACATGTACTCCTGC

AGATTTAGCCGGCAGTGCGTGGTGGACAAAGACAAGAGGAACCAGTGCCGCTACT

GCAGGCTCAAGAAATGCTTCCGGGCTGGCATGAAGAAGGAAGCCGTCCAGAATGA

GCGGGACCGGATCAGCACTCGAAGGTCAAGCTATGAGGACAGCAGCCTGCCCTCC

Hepatocyt ATCAATGCGCTCCTGCAGGCGGAGGTCCTGTCCCGACAGATCACCTCCCCCGTCTCC

H242D e nuclear GGGATCAACGGCGACATTCGGGCGAAGAAGATTGCCAGCATCGCAGATGTGTGTG factor 4 AGTCCATGAAGGAGCAGCTGCTGGTTCTCGTTGAGTGGGCCAAGTACATCCCAGCT

TTCTGCGAGCTCCCCCTGGACGACCAGGTGGCCCTGCTCAGAGCCCATGCTGGCGA

GCACCTGCTGCTCGGAGCCACCAAGAGATCCATGGTGTTCAAGGACGTGCTGCTCC

Kl TAGGCAATGACTACATTGTCCCTCGGCACTGCCCGGAGCTGGCGGAGATGAGCCGG

GTGTCCATACGCATCCTTGACGAGCTGGTGCTGCCCTTCCAGGAGCTGCAGATCGA

TGACAATGAGTATGCCTACCTCAAAGCCATCATCTTCTTTGACCCAGATGCCAAGG

GGCTGAGCGATCCAGGGAAGATCAAGCGGCTGCGTTCCCAGGTGCAGGTGAGCTTG

GAGGACTACATCAACGACCGCCAGTATGACTCGCGTGGCCGCTTTGGAGAGCTGCT

GCTGCTGCTGCCCACCTTG

Kl

VO oe

Kl

VO VO

Ul

© ©

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. name Sequence sequence sequence

CAGGCGTGACGCCAGTTCTAAATCTTGAAACAGAACAAAACTTCAAAGTACACCAA cgcggaattcctgtggcccagttcc ggccaagcttccaggttgaggttg

AATAGAACCTCCTTAAAGCATAAATCTCACGGAGGGTCTCGCCGCCAGTGGAAGGA agttc gcagg

GCCACCGCCCCCGCCCGACCATGGCCGAGGAGCTGGTCTTAGAGAGGTGTGATCTG

GAGCTGGAGACCAATGGCCGAGACCACCACACGGCCGACCTGTGCCGGGAGAAGC

TGGTGGTGCGACGGGGCCAGCCCTTCTGGCTGACCCTGCACTTTGAGGGCCGCAAC

TACGAGGCCAGTGTAGACAGTCTCACCTTCAGTGTCGTGACCGGCCCAGCCCCTAG

CCAGGAGGCCGGGACCAAGGCCCGTTTTCCACTAAGAGATGCTGTGGAGGAGGGT

GACTGGACAGCCACCGTGGTGGACCAGCAAGACTGCACCCTCTCGCTGCAGCTCAC

Tissue CACCCCGGCCAACGCCCCCATCGGCCTGTATCGCCTCAGCCTGGAGGCCTCCACTG

H274Ar transgluta GCTACCAGGGATCCAGCTTTGTGCTGGGCCACTTCATTTTGCTCTTCAACGCCTGGT minase GCCCAGCGGATGCTGTGTACCTGGACTCGGAAGAGGAGCGGCAGGAGTATGTCCTC

ACCCAGCAGGGCTTTATCTACCAGGGCTCGGCCAAGTTCATCAAGAACATACCTTG

GAATTTTGGGCAGTTTGAAGATGGGATCCTAGACATCTGCCTGATCCTTCTAGATGT

Ui CAACCCCAAGTTCCTGAAGAACGCCGGCCGTGACTGCTCCCGCCGCAGCAGCCCCG o TCTACGTGGGCCGGGTGTGGAGTGGCATGGTCAACTGCAACGATGACCAGGGTGTG

CTGCTGGGACGCTGGGACAACAACTACGGGGACGGCGTCAGCCCCATGTCCTGGAT

CGGCAGCGTGGACATCCTGCGGCGCTGGAAGAACCACGGCTGCCAGCGCGTCAAG

TATGGCCAGTGCTGGGTCTTCGCCGCCGTGGCCTGCACAGTGCTGAGGTGCCTGGG

CATCCCTACCCGCGTCG

Ui o κ

Ui o

Ui

U o

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

GAAGTGACGCGAGGCGTAGCGGAAGTTACTGCAGCGGCGGTGTTGTGCTGTGGGG cgcgggatcctgtggtgttcaaaac ggccaagcttttcagtgaagccat

AAGGGAGAAGGATTTGTAAACCCCGGAGCGAGGTTCTGCTTACCCGAGGCCGCTGC ggaattg ttggagt

TGTGCGGAGACCCCCGGGTGAAGCCACCGTCATCATGTCTGACCAGGAGGCAAAAC

CTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATATTAAACTCAAAGTC

ATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAACACATCTCAA

GAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACTCAGGT

TTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAATG

Ubiquitin- GAGGAAGAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAG homology

H292D domain GTTCTTTTGTAATGTGGTGTTCAAAACGGAATTGAAAACTGGCACCCCATCTCTTTG protein AAACATCTGGTAATTTGAATTCTAGTGCTCATTATTCATTATTGTTTGTTTTCATTGT

PIC1 GCTGATTTTTGGTGATCAAGCCTCAGTCCCCTTCATATTACCCTCTCCTTTTTAAAAA

TTACGTGTGCACAGAGAGGTCACCTTTTTCAGGACATTGCATTTTCAGGCTTGTGGT

Ui GATAAATAAGATCGACCAATGCAAGTGTTCATAATGACTTTCCAATTGGCCCTGAT o GTTCTAGCATGTGATTACTTCACTCCTGGGACTGTGACTTTCAGTGGGAGATGGAAG

TTTTTCAGAGAACTGAACTGTGGAAAAATGACCTTTCCTTAACTTGAAGCTACTTTT

AAAATTTGAGGGTCTGGACCAAAAGAAGAGGAATATCAGGTTGAAGTCAAGATGA

CAGATAAGGTGAGAGTAATGACTAACTCCAAAGATGGCTTCACTGAAGAAAAGGC

ATTTTAAGA

Ui o

-4

Ui o ∞

Ui o

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CAGAGTTGAGAATGGAGAGAATGTTACCTCTCCTGGCTCTGGGGCTCTTGGCGGCT cgcgggatccaccttacttccggga ggccaagctttgggattggtgactt

GGGTTCTGCCCTGCTGTCCTCTGCCACCCTAACAGCCCACTTGACGAGGAGAATCTG cgagg gctca

ACCCAGGAGAACCAAGACCGAGGGACACACGTGGACCTCGGATTAGCCTCCGCCA

ACGTGGACTTCGCTTTCAGCCTGTACAAGCAGTTAGTCCTGAAGGCCCTTGATAAG

AATGTCATCTTCTCCCCACTGAGCATCTCCACCGCCTTGGCCTTCCTGTCTCTGGGG

GCCCATAATACCACCCTGACAGAGATTCTCAAGGCCTCGAGTTCACCTCACGGAGA

CTTACTGAGGCAGAAATTCACTCAGAGCTTCCAGCACCTCCGCGCACCCTCAATCA

GTTCCAGCGATGAGCTGCAGCTGAGTATGGGAAATGCCATGTTTGTCAAAGAGCAA

Alpha-1 CTCAGTCTGCTGGACAGGTTCACGGAGGATGCCAAGAGGCTGTATGGCTCCGAGGC

H308D antichymo CTTTGCCACTGACTTTCAGGACTCAGCTGCAGCTAAGAAGCTCATCAACGACTACG trypsin TGAAGAATGGAACTAGGGGGAAAATCACAGATCTGATCAAGGACCCCGACTCGCA

GACAATGATGGTCCTGGTGAATTACATCTTCTTTAAAGCCAAATGGGAGATGCCCT

TTGACCCCCAAGATACTCATCAGTCAAGGTTCTACTTGAGCAAGAAAAAGTGGGTA

Ui ATGGTGCCCATGATGAGTTTGCATCACCTGACTATACCTTACTTCCGGGACGAGGA o GCTGTCCTGCACCGTGGTGGAGCTGAAGTACACAGGCAATGCCAGCGCACTCTTCA

TCCTCCCTGATCAAGACAAGATGGAGGAAGTGGAAGCCATGCTGCTCCCAGAGACC

CTGAAGCGGTGGAGAGACTCTCTGGAGTTCAGAGAGATAGGTGAGCTCTACCTGCC

AAAGTTTTCCATCTCGAGGGACTATAACCTGAACGACATACTTCTCCAGCTGGGCA

TTGAGGAAGCCTTCAC

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

GGAATTCCGTGGCCGGGACTTTGCAGGCAGCGGCGGCCGGGGGCGGAGCGGGATC cgcgggatcccaggtggggaggc ggccctcgagcagagcagtggg

GAGCCCTCGCCGAGGCCTGCCGCCATGGGCCCGCGCCGCCGCCGCCGCCTGTCACC tttgg aggca

CGGGCCGCGCGGGCCGTGAGCGTCATGGCCTTGGCCGGGGCCCCTGCGGGCGGCCC

ATGCGCGCCGGCGCTGGAGGCCCTGCTCGGGGCCGGCGCGCTGCGGCTGCTCGACT

CCTCGCAGATCGTCATCATCTCCGCCGCGCAGGACGCCAGCGCCCCGCCGGCTCCC

ACCGGCCCCGCGGCGCCCGCCGCCGGCCCCTGCGACCCTGACCTGCTGCTCTTCGC

CACACCGCAGGCGCCCCGGCCCACACCCAGTGCGCCGCGGCCCGCGCTCGGCCGCC

CGCCGGTGAAGCGGAGGCTGGACCTGGAAACTGACCATCAGTACCTGGCCGAGAG

CAGTGGGCCAGCTCGGGGCAGAGGCCGCCATCCAGGAAAAGGTGTGAAATCCCCG

H311Ar E2F-1 GGGGAGAAGTCACGCTATGAGACCTCACTGAATCTGACCACCAAGCGCTTCCTGGA

GCTGCTGAGCCACTCGGCTGACGGTGTCGTCGACCTGAACTGGGCTGCCGAGGTGC

TGAAGGTGCAGAAGCGGCGCATCTATGACATCACCAACGTCCTTGAGGGCATCCAG

CTCATTGCCAAGAAGTCCAAGAACCACATCCAGTGGCTGGGCAGCCACACCACAGT

GGGCGTCGGCGGACGGCTTGAGGGGTTGACCCAGGACCTCCGACAGCTGCAGGAG

AGCGAGCAGCAGCTGGACCACCTGATGAATATCTGTACTACGCAGCTGCGCCTGCT

CTCCGAGGACACTGACAGCCAGCGCCTGGCCTACGTGACGTGTCAGGACCTTCGTA

GCATTGCAGACCCTGCAGAGCAGATGGTTATGGTGATCAAAGCCCCTCCTGAGACC

CAGCTCCAAGCCGTGGACTCTTCGGAGAACTTTCAGATCTCCCTTAAGAGCAAACA

AGGCCCGATCGATGTTTTCC

U κ

U uι

Table 1

NumeriArray Left PCR primer Right PCR primer cal ref. Sequence name sequence sequence

CCACCATATCGGTCCCGTATTTCACATTGATAAGGTCCTGTTTCATTTCTCGTGACA gaacacacctttatggctggggctct ctctcttcagaggcatccaggaca

TTGGGTAGAATGAGGATCCTGTTTTCAATGGGTCGCTTTACCCTGGGACTGACAGG c gg

GAGGCTCTGACCATTTAGCCACCAAATGTAGGTGTAGTTCTCACTCTTAGGTTCACC

CCGCGGCCGATCGTCCCCCATACCTCGGCCATGCGGCCCCTGCTGCTACTGGCCCTG

CTGGGCTGGCTGCTGCTGGCCGAAGCGAAGGGCGACGCCAAGCCGGAGGACAACC

TTTTAGTCCTCACGGTGGCCACTAAGGAGACCGAGGGATTCCGTCGCTTCAAGCGC

TCAGCTCAGTTCTTCAACTACAAGATCCAGGCGCTTGGCCTAGGGGAGGACTGGAA

TGTGGAGAAGGGGACGTCGGCAGGTGGAGGGCAGAAGGTCCGGCTGCTGAAGAAA

Lysyl GCTCTGGAGAAGCACGCAGACAAGGAGGATCTGGTCATTCTCTTCACAGACAGCTA

H462B hydroxy la TGACGTGCTGTTTGCATCGGGGCCCCGGGAGCTCCTGAAGAAGTTCCGGCAGGCCA se GGAGCCAGGTGGTCTTCTCTGCTGAGGAGCTCATCTACCCAGACCGCAGGCTGGAG

ACCAAGTATCCGGTGGTGTCCGATGGCAAGAGGTTCCTGGGCTCTGGAGGCTTCAT

CGGTTATGCCCCCAACCTCAGCAAACTGGTGGCCGAGTGGGAGGGCCAGGACAGC

U GACAGCGATCAGCTGTTTTACACCAAGATCTTCTTGGACCCGGAGAAGAGGGAGCA

GATCAATATCACCCTGGACCACCGCTGCCGTATCTTCCAGAACCTGGATGGAGCCT

TGGATGAGGTCGTGCTCAAGTTTGAAATGGGCCATGTGAGAGCGAGGAACCTGGCC

TATGACACCCTCCCGGTCCTGATCCATGGCAACGGGCCAACCAAGCTGCAGTTGAA

CTACCTGGGCAACTACATCCCGCGCTTCTGGACCTTCGAAACAGGCTGCACCGTGT

GTGACGAAGGCTTGCG

Ui -4

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CGGGAGAGCGCGCTCTGCCTGCCGCCTGCCTGCCTGCCACTGAGGGTTCCCAGCAC acagacacacgatgttgtcaaggat agaacaaaggggagggtgaaga

CATGAGGGCCTGGATCTTCTTTCTCCTTTGCCTGGCCGGGAGGGCCTTGGCAGCCCC gg aagg

TCAGCAAGAAGCCCTGCCTGATGAGACAGAGGTGGTGGAAGAAACTGTGGCAGAG

GTGACTGAGGTATCTGTGGGAGCTAATCCTGTCCAGGTGGAAGTAGGAGAATTTGA

TGATGGTGCAGAGGAAACCGAAGAGGAGGTGGTGGCGGAAAATCCCTGCCAGAAC

CACCACTGCAAACACGGCAAGGTGTGCGAGCTGGATGAGAACAACACCCCCATGT

GCGTGTGCCAGGACCCCACCAGCTGCCCAGCCCCCATTGGCGAGTTTGAGAAGGTG

SPARC

TGCAGCAATGACAACAAGACCTTCGACTCTTCCTGCCACTTCTTTGCCACAAAGTGC (secreted

ACCCTGGAGGGCACCAAGAAGGGCCACAAGCTCCACCTGGACTACATCGGGCCTTG

H467A protein acidic and CAAATACATCCCCCCTTGCCTGGACTCTGAGCTGACCGAATTCCCCCTGCGCATGCG rich in GGACTGGCTCAAGAACGTCCTGGTCACCCTGTATGAGAGGGATGAGGACAACAAC cysteine) CTTCTGACTGAGAAGCAGAAGCTGCGGGTGAAGAAGATCCATGAGAATGAGAAGC

GCCTGGAGGCAGGAGACCACCCCGTGGAGCTGCTGGCCCGGGACTTCGAGAAGAA

Ui CTATAACATGTACATCTTCCCTGTACACTGGCAGTTCGGCCAGCTGGACCAGCACCC

∞ CATTGACGGGTACCTCTCCCACACCGAGCTGGCTCCACTGCGTGCTCCCCTCATCCC

CATGGAGCATTGCACCACCCGCTTTTTCGAGACCTGTGACCTGGACAATGACAAGT

ACATCGCCCTGGATGAGTGGGCCGGCTGCTTCGGCATCAAGCAGAAGGATATCGAC

AAGGATCTTGTGATCTAAATCCACTCCTTCCACAGTACCGGATTCTCTCTTTAACCC

TCCCCTTCGTGTTTCC

Table 1

NumeriArray Left PCR primer Right PCR primer

Sequence cal ref. name sequence sequence

CACGCTTGCCGCCGCCCCGCAGAAATGCTTCGGTTACCCACAGTCTTTCGCCAGATG tttcagatggagtggctgtgctga ttgcacccattccagggtcctt

AGACCGGTGTCCAGGGTACTGGCTCCTCATCTCACTCGGGCTTATGCCAAAGATGT

AAAATTTGGTGCAGATGCCCGAGCCTTAATGCTTCAAGGTGTAGACCTTTTAGCCG

ATGCTGTGGCCGTTACAATGGGGCCAAAGGGAAGAACAGTGATTATTGAGCAGGG

TTGGGGAAGTCCCAAAGTAACAAAAGATGGTGTGACTGTTGCAAAGTCAATTGACT

TAAAAGATAAATACAAGAACATTGGAGCTAAACTTGTTCAAGATGTTGCCAATAAC

ACAAATGAAGAAGCTGGGGATGGCACTACCACTGCTACTGTACTGGCACGCTCTAT

Heat AGCCAAGGAAGGCTTCGAGAAGATTAGCAAAGGTGCTAATCCAGTGGAAATCAGG shock AGAGGTGTGATGTTAGCTGTTGATGCTGTAATTGCTGAACTTAAAAAGCAGTCTAA

H572A protein 60 ACCTGTGACCACCCCTGAAGAAATTGCACAGGTTGCTACGATTTCTGCAAACGGAG (Chaperon ACAAAGAAATTGGCAATATCATCTCTGATGCAATGAAAAAAGTTGGAAGAAAGGG in) TGTCATCACAGTAAAGGATGGAAAAACACTGAATGATGAATTAGAAATTATTGAAG

GCATGAAGTTTGATCGAGGCTATATTTCTCCATACTTTATTAATACATCAAAAGGTC

U AGAAATGTGAATTCCAGGATGCCTATGTTCTGTTGAGTGAAAAGAAAATTTCTAGT vo ATCCAGTCCATTGTACCTGCTCTTGAAATTGCCAATGCTCACCGTAAGCCTTTGGTC

ATAATCGCTGAAGATGTTGATGGAGAAGCTCTAAGTACACTCGTCTTGAATAGGCT

AAAGGTTGGTCTTCAGGTTGTGGCAGTCAAGGCTCCAGGGTTTGGTGACAATAGAA

AGAACCAGCTTAAAGATATGGCTATTGCTACTGGTGGTGCAGTGTTTGGAGAAGAG

GGATTGACCCTGAATC

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Table 3

(+)-cis-3,5-dimethyl-2-(3- benzoyl peroxide pyridyl)thiazolodin-4 benztropine

(s)-warfarin berylium

1 ,2-Dibromomethane beta carotene

1 -chloro-2-nitrobenzene betamethasone

2,4-dinitrophenol betamethasone valerate

2,4-dinitrotoluene bethanechol

2-acetylaminofluorene biphenyl

2-azido-2-deoxycytidine bisacodyl

2-methylpentane bismuth subsalicylate plus

3-methylpentane bisoprolol/HCTZ

4,4'-methylene bis bleomycin

4-acetamidofluorene bradykinin antagonist

5-azacytidine bromfenac

5-chlorouracil brominide tartrate

5-fluorouracil bromobenzene

6-mercaptopurine bromocriptine

7, 12-dimethylbenz[a]anthracene bronchodilators acetaminophen bta-napthylamine acetaminophen codeine buclizine acetohydroxamic acid budesonide acetone bumetanide acetylsalicylic acid bupropion HCL acridine buspirone acrylamide busulfan acrylonitrile cadmium actinomycin cadmium chloride actinomycin D caffeine acyclovir calcipotriene adenosine calcitonin salmon aflatoxin Bl calfactant albuterol camptothecin alendronate candesartan cilexetil alendronate sodium capsaicin alglucerase captopril allopurinol carbamate(s) allyl alcohol carbamazapine alosetron carbaryl alprazolam carbenicillin alprostadil carbidopa alteplase carbon disulfide aluminum carbon monoxide ambenonium carbon tetrachloride amifostine carboplatin amiloride carisoprodol aminobenzoate potassium carmustine aminoglutethimide carvedilol benzene cefaclor benzidine cefepime benzo(a)pyrene ce rozil benzodiazepines ceftibuten cefuroxime cyanides celecoxib cyclacillin cephalexin cyclandelate cephalosporins cyclizine cerivastatin cyclobenzaprine cetirizine cyclohexane chenodiol cyclohexanone chlophedianol cycloheximide chloral hydrate cyclopegic chlorambucil cyclopentolate chloramphenicol cyclophosphamide chloroform cycloserine chloroquine cyclosporin A chlorpromazine cyclosporine chlorpropamide cyclosporine A chlorthalidone cytosine arabinoside chlorzoxazone cytoxin cholestyramine dacarbazine chromium VI dalteparin injection cimetidine danazol cinoxacin dantrolene ciprofloxacin dapsone cisapride daunomycin cisplatin daunorubicin citalopram DDT clarithromycin DEHP clavulanate dehydrocholic acid clavulanic acid desmopressin clenbuterol desogestrel clidinium dexamethasome clindamycin dextromethorphan clofibrate dextrothyroxine clomiphene diazepam clonazepam diazoxon clonidine dichloralphenazone clotrimoxazole dichlorobenzene cloxacillin dichloromethane clozapine diclofenac

CMC diclofenacdihydrazine cobalt dicloxacillin codeine dicyclomine colchicine didanosine colestipol dieldrin collagen-alginate diethylamine conjugated estrogens diethylhexylphthalate copolymer- 1 diethylstilbestrol copper diethystilbesterol corticosterone difenoxin cortisone diflunisal courmarin digitalis glycosides cromolyn digitoxin cumene digoxin cyanamide dihydrazine dihydroergotamine mesylate ethyleneglycol(s) dihydrolazine etidronate diltiazem etoposide dimethyl sulfoxide etretinate dimethylacetamide exemestane dimethylformamide famciclovir dimethylhydrazine famotidine dimethylnitrosamine felbamate dinitroorthocresol felodipine dinoprostone felodipine SR dione fenofϊbrate dioxane fenoldopam mesylate diphenidol fentanyl citrate diphenoxylate fexofenadine dipyridamole fialuridine disopyramide fϊnasteride disulfiram flavoxate divalproex flecainide acetate divalproex sodium flosequinan

DMBA fluconazole docusate sodium flunisolide dolasetron mesylate fluoride donepezil fluoroquinolones doxazosin fluorouracil doxercalciferol fluoxetine doxorubicin flutamide doxycycline fluticasone enalapril fluticasone propionate endotoxin fluvastatin endrin fluvoxamine maleate enflurane foscarnet sodium enoxaparin fosinopril entacapone fosphenytoin ephedrine furazolidone epirubicin furfural eptifϊbatide furfuryl alcohol ergoloid mesylates furosemide ergonovine gabapentin erythromycin ganciclovir erythromycin estolate ganirelix acetate estradiol gemcitabine estramustine gemfϊbrozil etanercept gentamicin ethacrynic acid germanium ethanol glimepiride ethchlorvynol glipizide ethinamate glucagon ethinyl estradiol glyburide ethionamide glycopyrrolate ethyl methanesulfonate gold compounds ethylbenzene gold sodium thiomalate ethylene oxide granisetron ethyleneglycol dinitrate grepafloxacin griseofulvin itraconazole guaifenesin kanamycin guanabenz ketoconazole guanadrel ketorolac guanethidine lactulose guanfacine lamivudine, 3TC guanine lamotrigine haloperidol lansoprazole halothane latamoxef heparin latanoprost hexachlorobenzene lead hexachlorobutadiene lead tetraethyl hismanol leflunomide hydantoin letrozole hydralazine leucovorin hydrochlorothiazide leuprolide hydrocodone levamisole hydrocortisone levetiracetam hydroxychloroquine levobupivacaine hydroxyurea levocabastine hydroxyzine levocarnitine hyoscine levodopa hyoscyamine levofloxacin hyperozia levonorgestrel ibuprofen levothyroxine ibutilide fumarate lidocaine imiglucerase injection lincomycin imiquimod 5% cream lindane inactivated hepatitis A vaccine lipopolysaccharide indapamide liposomal amphotericin B indinavir lisinopril indomethacin lispro insulin insulin lithium interferon-beta-la (recombinant) 1-norgestrel interferon-beta-lb (recombinant) 1-norgestrel/ethinyl estradiol iodinated glycerol lomustine iodoacetamide loperamide iodoquinol loracarbef ipecac loratadine iphosphamide Loratidine/Pseudoephedrine ipratropium lorazepam irbesartan losartan irinotecan lovastatin isometheptene loxapine isoniazid magnesium sulfate isonicotinic acid maleic anhydride isopropanol manganese isopropylnitrate maprotiline isoproterenol masoprocol isosorbide mononitrate S.A. mazindol isotretinoin mecamylamine isoxsuprine mechlorethamine isradipine meclizine medroxyprogesterone nafarelin mefloquine nafcillin melatonin nalidixic acid melphalan naloxone menotropin naltrexone meprobamate naproxen merbarone naratriptan mercaptopurine natamycin mercury navirapine meropenem nedocromil mesalamine nefazodone metformin neomycin methanol neomycin Polymx/HC methenamine neostigmine methicillin N-hexane methotrexate nicardipine methyl methanesulfonate nickel methylcellulose nicorandil methylchloride nicotine methyldopa nifedipine methylergonovine nimodipine methylethylketone nitrobenzene methylmercury nitrofurantoin methylphenidate nitroglycerin methylprednisolone nitroglycerine methyprylon nitrous oxide methysergide nizatidine metoclopramide N-nitroso-N-ethylurea metoprolol N-nitroso-N-methylurea metronidazole norethindrone metyrapone norethindrone/ ethinyl estradiol metyrosine norgestimate mexiletine norgestimate/ethinyl estradiol mibefradil norgestrel miconazole cream 2% norgestrel/ethinyl estradiol miglitol nylidrin minocycline nystatin minoxidil ofloxacin misoprostol oligomycin mitomycin C olsalazine mitotane omeprazole mitoxantrone Organophosphorus mixed amphetamines orphenadrine moclobemide o-toluidine molindone oxacillin mometasone oxaprozin monobromomethane oxtriphylline monochlorobenzene oxybutynin moricizine oxycodone moxifloxacin oxymetazoline mupirocin paclitaxel nabilone pancreatin nabumetone pancrelipase papaverme pnmaqume paraldehyde primethamine paramethasone primidone parathione probenecid paregoric probucol paroxetine procainamide pediculisides procarbazine pemoline proflavin penicillamine progesterone penicillin progestins pentachlorophenol promethazine pentamidine propafenone pentoxifylline propantheline pepsin propoxyphene pergolide propranolol perhexiline propulsid perindopril propyleneglycol perphenazine pseudoephedrine pexiganan acetate psoralens phenazopyridine psyllium phendimetrazine puromycin phenformin pyridostigmine phenobarbital pyridoxine (vitamin b-6) phenol quinacrine phenolphthalein quinapril phenothiazines quinidine phentermine quinine phenylephrine rabeprazole phenylhydrazine HCL raloxifene phenylpropanolamine ramipril phenytoin ranitidine phorbol 12-myristate 13-acetate recombinant clotting factor VHI diester recombinant interferon alpha-2b phtalic anhydride recombinant OspA pilocarpine remoxipide pioglitazone reserpine piroxicam rezulin podophyllum ribavirin poloxamer 188 rifampicin polycarbophil calcium rifampin polychlorinated biphenyl rimantadine polycyclic hydrocarbons risedronate polyethylene glycol risperidone polythiazide ritodrine potassium chloride rosiglitazone potassium iodide salicylates potassium phosphates salmeterol pramipexole saquinavir pravastatin scopolamine prazosin seldane prednisolone selegiline prednisone selenium pregnenolone- 16-alpha-carbonitrile sertraline sibutramine timolol sildenafϊl citrate tiopronin silver tirofiban simethicone tobramycin simvastatin tobramycin/dexamethasone s-mephenytoin tocainide sodium azide tolbumamide sodium ferric gluconate tolcapone soman tolterodine somatostatin toluene sotalol toluene diisocyanate spironolactone topotecan stanol esters toremifene streptozotocin tramadol stryrene trandolapril succinimide transplatin sucralfate trastuzumab sulfacytine trazodone sulfadoxine tretinoin sulfamethoxazole triamcinolone sulfasalazine triamterene/HCTZ sulfinpyrazone triamterine sulfisoxazole triazolam sumatriptan trichloroethane synthetic pyrethroids trichloroethylene tacrine triethylamine tamoxifen triethylbenzenes tamsulosin triethylenemelamine

T-butylhydroperoxide triethylenethiophosphoramide

TCDD trihexyphenidyl tellerium trilostane telmisartan trimeth/sulfameth temazepam trimethobenzamide terazosin trimethoprim terbinafϊne HCl troglitazone terbutaline sulfate trovafloxacin terfenadine uranium terpin hydrate urokinase tert-butylphenol ursodiol testolactone valproic acid tetrachloroethylene valsartan tetracycline vanadium tetracycline HCl vancomycin thalium venlafaxine theophylline verapamil thiamine vincristine thiazide vinyl chloride thioguanine warfarin thiopurine Wy 14,643 thiothixene xanthine tiagabine xylene ticlopidine xylometazoline tienilic acid zafϊrlukast zalcitabine zinc zidovudine zolpidem

Table 4

Human In Vitro Hepatocyte microarray data

• Up-regulated genes:

-Activating transcription factor 3 (amiodarone, chlorpromazine, paracetamol)

—Activating transcription factor 4 (amiodarone, chlorpromazine, paracetamol)

-BRCA1 (tacrine, perhexiline)

-Phenol sulfotransferase (amiodarone, chlorpromazine)

-Hepatocyte nuclear factor 4 (amiodarone, paracetamol)

-Thioredoxin (chlorpromazine, paracetamol, perhexiline)

- Ferritin H-chain (chlorpromazine, paracetamol, perhexiline, amiodarone) -Gadd 153 (amiodarone, chlorpromazine)

- Insulin-like growth factor binding protein 1 (chlorpromazine, paracetamol, perhexiline, amiodarone)

-T-cell cyclophilin (chlorpromazine, paracetamol)

-Cathepsin B (chlorpromazine, paracetamol, perhexiline, amiodarone, tacrine)

-Cathepsin L (amiodarone, paracetamol)

• Down-regulated genes:

-Cytochrome c oxidase subunit II (amiodarone, chlorpromazine, perhexiline) -Gamma-glutamyl transpeptidase (amiodarone, paracetamol) -Glyceraldehyde 3 -phosphate dehydrogenase (paracetamol, amiodarone, chlorpromazine, perhexiline, tacrine)

-Apolipoprotein CHI (amiodarone, chlorpromazine, paracetamol)

-Fas antigen (amiodarone, paracetamol)

-Sterol carrier protein 2 (amiodarone, tacrine)

-Transferrin (amiodarone, chlorpromazine, paracetamol, tacrine)

-Tyrosine aminotransferase (paracetamole, amiodarone, tacrine) Table 5

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