WO2006138716A2 - Thermodynamically defined gene specific primers - Google Patents

Abstract

A method is described for designing an optimized nucleic acid primer for reverse transcription of an RNA target of interest, comprising selecting a region of the RNA target for designing a target-specific primer, and optimizing the kinetics of hybridization of candidate target-specific primers to the RNA, thereby designing an optimized target-specific RT primer. The RT primers can be about 7-9 nt in length and are useful, for example, for formalin fixed paraffin embedded tissue samples or for other compromised samples. Primers designed by the method, RT-PCR methods using such primers, and kits containing the primers are presented.

Description

Thermodynamically Defined Gene Specific Primers

FIELD OF THE INVENTION

The present invention relates, e.g., to a method for designing an optimized nucleic acid primer for reverse transcribing RNA, primers designed by such a method, RT-PCR methods using such primers, and kits containing the primers.

BACKGROUND INFORMATION

Formalin-fixed, paraffin-embedded tissues (PET) are a useful source of research material with the potential of providing biological information in conjunction with known clinical outcome. For example, archived tissues from cancer patients can serve as a potential gold mine of information when linked to underlying expression profiles, and can therefore serve to validate and test biomarkers associated with cancer and to identify therapeutic targets. Furthermore, PET tissue samples are useful for diagnostic assays. For example, a PET sample from a cancer patient can be used to diagnose the type and/or degree of malignancy of the cancer.

The analysis of transcription patterns obtained from PET samples is highly dependent on the efficiency of reverse transcription. Unfortunately, RNA isolated from PET samples is considered to be a poor material for molecular analyses, since the RNA is frequently degraded to fragments of about 30-200 nucleotides (nt) by endogenous and exogenous ribonucleases (RNase). In addition, the RNA, in particular the poly(A) tails of mRNA, is often chemically modified, making it a poor template for cDNA synthesis, e.g. synthesis primed with oligo(dT) primers.

Tissues such as autopsy materials or samples from crime scenes behave in much the same manner as PET samples, because of degradation of cell constituents due to exposure to environmental insults. The degradation produces molecular fragmentation of nucleic acids and cellular components. Such degraded tissues are sometimes referred to herein as compromised tissues. One type of forensic investigation is to identify the tissue from which a sample obtained at a crime scene originated, by characterizing RNA transcription of tissue-specific markers within the sample. The ability to obtain results from compromised tissues can aid in the reconstruction of a crime scene.

The present inventors have developed a method for designing primers for reverse transcription, which can then be subjected to amplification procedures such as RT-PCR, for PET or other compromised samples.

DESCRIPTION OF THE INVENTION

The present invention relates, e.g., to a method for designing an optimized nucleic acid primer for reverse transcription (RT) of an RNA target of interest, comprising selecting a region of the RNA target for designing a target-specific primer, and optimizing the kinetics of hybridization of candidate target-specific primers corresponding to that region of the RNA, thereby designing an optimized target-specific RT primer.

The inventors have recognized advantages of designing gene-specific RT primers based on the thermodynamics of hybridization of the primer to the template to be reversed transcribed. For example, in the case of a DNA primer for reverse transcription of an mRNA of interest, a gene-specific primer (GSP) is designed by identifying a suitable region of the mRNA, then designing a DNA RT primer corresponding to that region, based on, among other factors, the temperature of the hybridization reaction between the RT primer and the RNA template, and the T_m of the RNA/DNA hybrid. Surprisingly, for most sequences, depending on the G+C content, the annealing temperature and other factors, the optimum primer length for such a primer is often about 7-9 nucleotides, e.g., 8-9 nt. This is smaller than gene specific primers which are generally used for reverse transcription (Le., primers designed on the basis of the T_n, of a DNA/DNA hybrid). The Examples herein demonstrate that by using RT primers designed according to the invention, the reverse transcription reaction is optimized and proper specificity is observed, without mis-priming. When cDNA generated with such primers is subsequently amplified, e.g. by PCR, the level of amplified product is significantly higher than when RT primers designed by conventional methods are used. The method is also applicable to primers other than DNA primers, including LNA (linked nucleic acid) or RNA primers. In those cases, primers are designed taking into account the hybridization kinetics of an RNA template to a primer having the particular nucleic acid composition.

Advantages of the methods of the invention include low levels of improper priming and the resultant background of undesired products, such as occurs following random priming of an rnRNA target; the ability to conduct reverse transcription even in the presence of modified or degraded poly(A) tails; sensitive signal detection; and prevention of primer-dimer formation. The inventive method allows for highly specific cDNA synthesis and sensitive detection of low abundance or degraded RNA transcripts, such as RNA from PET samples or compromised samples for forensic analysis. Multiple gene-specific primers may be present in a single reaction.

One aspect of the invention is a method for designing an optimized nucleic acid primer for reverse transcription of an RNA target of interest, comprising selecting a region of the RNA target for designing a target-specific primer, and optimizing the kinetics of hybridization of candidate target-specific primers corresponding to that region of the RNA, thereby designing an optimized target-specific RT primer.

In embodiments of this method, the RNA is an mRNA; and/or the primer is DNA, LNA or RNA. In a preferred embodiment, the primer is DNA, the kinetics of hybridization are optimized for a hybridization temperature of about 37ºC to about 42ºC; and/or the optimized primer is about 7-9 nucleotides (nt), e.g. 8-9 nt, in length.

Another aspect of the invention is an optimized nucleic acid RT primer designed by a method of the invention; or a set of RT primers of the invention, wherein the set contains no more than about 200 RT primers.

Another aspect of the invention is a method for reverse transcribing one or more RNA targets of interest, in a reaction mixture, comprising hybridizing optimized nucleic acid RT primers designed according to a method of the invention to the RNA, and reverse transcribing the RNA to generate target-specific DNA product(s), wherein the number of RT primers in the reaction mixture is no more than about 4-5 times greater than the number of RNA targets.

Another aspect of the invention is a method for reverse transcribing one or more RNA targets of interest, in a reaction mixture, comprising hybridizing target-specific primers which are about 7-9 nt (e.g., 8-9 nt) in length to the RNA, and reverse transcribing the RNA to generate target-specific DNA products, wherein the number of RT primers in the reaction mixture is no more than about 4-5 times greater than the number of RNA targets.

Another aspect of the invention is an amplification method, comprising amplifying a target-specific DNA product of the invention. In one embodiment, the amplification method is PCR (polymerase chain reaction).

Another aspect of the invention is a method for detecting expression of a marker of interest in a cell or tissue, comprising reverse transcribing RNA from the cell or tissue by a method of the invention to produce one or more marker-specific DNA products; and amplifying the marker-specific DNA product(s). The presence of an amplification product indicates expression of the marker in the cell or tissue. In a preferred embodiment, the amplification is by PCR. Preferably, the PCR is carried out using a marker-specific forward PCR primer and a marker-specific reverse PCR primer, wherein the reverse PCR primer comprises the RT primer. In embodiments of the invention, the reverse transcription and amplification are earned out on RNA which is degraded, e.g. to fragments about 60-200 nucleotides in length, and/or the RNA is from a formalin-fixed, paraffin-embedded tissue or a tissue for forensic or diagnostic analysis. In one embodiment, the marker of interest is a marker whose over-expression or under-expression is associated with the presence of a cancer (i.e., a cancer marker).

Another aspect of the invention is a kit for detecting an RNA target of interest, comprising a nucleic acid RT primer of the invention and, optionally, suitable PCR primers.

Another aspect of the invention is a method for generating a set of optimized nucleic acid primers corresponding to multiple sites on an mRNA expressed from a target of interest, comprising designing a first optimized nucleic acid RT primer from a first region of the RNA target by a method of the invention; designing a second optimized nucleic acid RT primer from a second region, about 100-300 nt away from the first primer, by a method of the invention; and repeating the process until RT primers are designed corresponding to multiple sites on the RNA.

Methods of the invention may be adapted to high throughput methodology. For example, amplification products, such as PCR-generated DNA products, of the invention may be contacted with an array of probes corresponding to multiple markers, such as markers for cancers, and screened for the presence and/or degree of expression of those markers.

This invention relates, e.g., to a method for designing an optimized nucleic acid primer for reverse transcription of an RNA target of interest (an RT primer), comprising selecting a region of the RNA target for designing a target-specific primer, and optimizing the kinetics of hybridization of candidate target-specific primers corresponding to that region of the RNA, thereby designing an optimized target-specific RT primer. As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the reference above to "an" optimized RT primer includes multiple optimized primers, which may be the same or mixtures of different primers. A "target-specific primer" (TSP) or "gene-specific ρrimer"(GSP) refers to a primer that specifically (preferentially) binds to (e.g., anneals to, hybridizes to, or is otherwise specifically associated with) a sequence in a nucleic acid from a particular target nucleic acid or a particular gene, or to a complement thereof. For example, the primer may bind to a target sequence with at least about twice the efficiency as it binds to a control.

Typical methods for designing optimized nucleic acid primers are presented in the Examples. Generally, one selects a region of the RNA target that is located at, or close to, the 3' end of the RNA. Candidate primers are chosen that correspond to (e.g., are specific for) the selected region. The candidate primers that "correspond" to the selected region contain sequences which are complementary to sequences within the selected region of the RNA. These complementary sequences can correspond to sequences in the entire region, or to a smaller set of sequences within the region. Generally, the primer sequences are completely complementary to sequences in the region. Candidate RT primers may also be designed to correspond to other regions of the target RNA. In some embodiments, as is discussed below, RT primers are designed that correspond to (are complementary to) several regions of a single RNA of interest.

The next step in the method is to analyze the kinetics of hybridization to the RNA of candidate primers corresponding to the selected region, at a chosen temperature, and to select an optimized primer from among the candidate primers. The chosen temperature is generally the temperature at which the reverse transcriptase functions optimally. Generally, this temperature lies between about 37ºC and about 42ºC. In ranges presented herein, such as temperature ranges or ranges of sizes of oligonucleotides, the end points are included within the range. In some embodiments of the invention, reverse transcriptase enzymes are used which function at temperatures other than about 37ºC to 42ºC, e.g. at higher temperatures. See, e.g., the discussion of reverse transcriptases that function at higher temperatures in chapter 16 ("Amplification of RNA: High temperature Reverse Transcription and DNA Amplification with a Magnesium-activated Thermostable DNA Polymerase") of Dieffenbach et al. PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, current edition. The length of the optimized primer may vary according to a number of factors, including the temperature of hybridization of the primer to the template; the nature of the backbone of the nucleic acid of the primer (e.g., whether it is DNA, RNA, PNA, LNA or combinations thereof, or the like); and/or the G+C content of the nucleotide sequence. In general, optimal DNA primers for reverse transcribing RNA targets at about 37ºC to about 42ºC have a length of about 7-9 nucleotides, which is smaller than primers that are generally used for reverse transcription (i.e., primers designed on the basis of the T_n, of a comparable DNA/DNA hybrid). The Examples show that, surprisingly, such optimized primers give rise to higher levels of amplified product than do the longer primers which are designed on the basis of the T_m of a DNA/DNA hybrid.

A nucleic acid primer that is specific for a target RNA of interest contains a sufficient number of contiguous nucleotides to be unique. To be unique, an RT primer of the invention must be of sufficient size to distinguish it from the complement of other expressed sequences. One way to estimate whether a primer is unique is to compare its sequence to the nucleotide sequences of expressed nucleic acids in computer databases, such as GenBank. Such comparative searches are standard in the art. In some cases, a 7-9-mer RT primer of the invention may, by chance, hybridize to a second, undesired, RNA sequence, in addition to the desired target RNA; in such a case, additional sequences that are present in the reverse PCR primer which comprises the RT primer will prevent further amplification of the unwanted cDNAs. Representative examples of RT primers of the invention include, but are not limited to, the primers shown in Table 4.

To calculate the melting temperature of candidate primers, in order to optimize primers for reverse transcription, well-known algorithms and computer programs are available which take into account salt concentrations, % GC content, nucleotide length or the like. Among the factors that can be taken into account are the composition of the oligo- or polynucleotide (length, base composition, type of duplex, sequence, and the extent of precise base pairing that occurs between the two strands) and the composition of the solvent (primarily the salt concentration and denaturants such as formamide) used in the reaction process. An example of a computer program for determining melting temperatures of DNA/DNA hybrids is Primer Express® software, available through ABI (Foster City, CA). With regard to algorithms for determining Tm's of RNA/DNA hybrids, RNA contains a hydroxyl group at the 2' carbon that is not present in DNA and which affects the natural helical turn rate of RNA and base stacking in the RNA/DNA hybrid. Algorithms taking into account these factors have been described for calculating T_m's of DNA/RNA hybrids in, e.g., Sugimota (1995) Biochemistry 34, 1121-1126 and Mathews et a!. (1999) RNA 5, 1458- 1469. Comparable calculations can be made for the T_m's of LNA/RNA hybrids or RNA/RNA hybrids, e.g. using nearest neighbor calculations. The optimal lengths for LNA and RNA RT primers designed to hybridize to RNA templates range from about 6-25 nts in length. For example, suitable, LNA primers can be about 12 nt, and RNA primers can be about 6-10 nt. A further routine option for any of the types of RT primers discussed above is to empirically calculate the melting temperature of a putative RT primer.

Another aspect of the invention is a method for extending annealed primers of the invention to generate target-specific nucleic acids (preferably target-specific DNAs). Typically the primers are extended in a sequence- specific manner. Extension of a primer in a sequence-specific manner includes any method wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence-specific manner, therefore, includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription or reverse transcription. Much of the discussion herein is directed to "RT primers," used in methods of reverse transcription. However, it is to be understood that the discussion of primers of the invention also applies to the use of the primers for any extension method, including those mentioned above. Techniques and conditions that amplify the primer in a sequence-specific manner are preferred. In preferred embodiments the primers are used to generate target-specific DNA products that are then used for DNA amplification reactions, such as PCR, or direct sequencing. It is to be understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.

A preferred embodiment is a method for reverse transcribing an RNA target of interest, comprising hybridizing (annealing) an optimized nucleic acid RT primer designed according to a method of the invention to the RNA target, and reverse transcribing the RNA to generate a target-specific DNA product. In one embodiment, the method comprises (a) designing an optimized nucleic acid RT primer by a method of the invention; (b) hybridizing the optimized target-specific primer to the RNA, under conditions effective for specific hybridization; and (c) reverse transcribing the RNA to generate a target-specific DNA product. Conditions "effective for" specific hybridization will be evident to the skilled worker. Such conditions include, e.g., suitable temperature, salts, etc. In embodiments of the invention, the RNA is an mRNA; the primer is DNA, LNA or RNA; the primer is DNA and the kinetics of hybridization are optimized for a hybridization temperature of about 37ºC to about 42ºC; and/or the optimized target-specific primer is about 7-9 nt in length (e.g., is 8-9 nt in length).

Another aspect of the invention is a method for reverse transcribing one or more RNA targets of interest, in a reaction mixture, comprising hybridizing target-specific primers which are about 7-9 nt {e.g., 8-9 nt) in length to the RNAs, and reverse transcribing the RNAs to generate target-specific DNA products. A "reaction mixture," as used herein, refers to a mixture comprising enzymes, substrates, primers, etc. in a suitable liquid (e.g., in a buffer containing suitable salts and/or other components). In this method, a limited number of RT primers is present in the reaction mixture. Generally, the number of RT primers is no more than about four to five times greater than the number of RNA targets. For example, no more than about 75, 100, 150, 200, 250 or 300 RT primers are present in the reaction mixture. This method differs from reverse transcription reactions in which random primers, such as random hexamer or random nonamer primers, are used to prime reverse transcription, at least because when randomly generated primers are used, tens to hundreds of thousands of the random primers are generally present in each reverse transcription reaction mixture.

Enzymes for reverse transcription are conventional and well-known in the art. Included are, e.g., M-MLV RT, Tth+ polymerase, Stratascript™ RT, AMV, and modified MMLV-RT. The GeneAmp® AccuRT RNA PCR enzyme is a designer DNA polymerase that enables Mg2+-activated, single-enzyme RT-PCR in a one-step, single- tube-reaction. Thermostable and thermoactive, the enzyme allows reverse transcription and PCR amplification to be performed at elevated temperatures, which helps to remove secondary structures from the template and results in highly stringent, accurate annealing of gene-specific primers

Any suitable RNA can serve as a target for reverse transcription by a method of the invention. For example, regulatory RNAs (antisense transcripts), which do not code for polypeptide, including naturally occurring siRNAs (small interfering RNAs) and srRNAs (small regulatory RNAs), can be reversed transcribed and analyzed by methods of the invention. In a preferred embodiment, the RNA target is an mRNA. Much of the discussion herein is directed to mRNA targets. However, it is to be understood that other types of RNA targets are also included.

Another aspect of the invention is a method for amplifying a target- specific nucleic acid product of the invention (e.g., a DNA product produced by a reverse transcription method of the invention). Any of a variety of well-known amplification procedures can be used. Preferably, PCR, or a variation thereof, is used. Other amplification methods include Q-Beta-replicase mediated amplification, ligation PCR, ligation-based strand displacement amplification, and NASBA (nucleic acid sequence based amplification).

A preferred amplification method is a PCR method that comprises amplifying a DNA product using a target-specific forward PCR primer and a target-specific reverse PCR primer, wherein the reverse PCR primer comprises the RT primer. Preferably, the RT primer is a truncated version of the target-specific reverse PCR primer. That is, the reverse PCR primer comprises the RT primer (e.g., a target-specific primer sequence of about 7-9 nts) on its 5¹ end, and further comprises additional target-specific nucleotides (e.g., at least about 8 - 19 additional target-specific nucleotides) on its 3' end. A reverse PCR primer will thus typically have at least about 15- 28 nucleotides, depending upon the specific nucleotide content of the sequence, the nature of the nucleic acid backbone, etc. Thus, a reverse PCR primer can have, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 additional target-specific nucleotides at its 3' end. The relationship between the RT primer and the reverse PCR primer as described herein provides an advantage to the RT and PCR primers. The preceding length calculations apply primarily to DNA primers used in PCR reactions. PCR primers composed of other nucleic acid backbones, such as RNA, PNA, LNA etc will of course be of different lengths, as will be evident to the skilled worker.

A truncated, gene-specific RT primer of this invention (the 5'- portion of a reverse PCR primer sequence) , when annealed to an RNA target, generally exhibits a melting temperature that is about 5ºC to about 20ºC lower than the melting temperature of the corresponding reverse PCR primer, when annealed to a DNA template. Thus, for example, the melting temperature of an RT primer/RNA template hybrid of the invention can be about 5°C, 6ºC, 7°C, 8°C, 9°C, 10ºC, HºC, 12ºC, 13°C, 14°C, 15°C, 16ºC, 17°C, 18°C, 19°C or 20ºC lower than the melting temperature of a corresponding PCR primer/DNA hybrid. In one embodiment, the melting temperature of an RT primer/RNA hybrid of the invention is from about 37°C to about 42ºC, and the corresponding melting temperature of a PCR primer/DNA hybrid is the typical melting temperature for a PCR primer, i.e. about 60ºC. It is contemplated that the 5' 7-9 nucleotides of any reverse PCR primer can be identified as a functional truncated, gene-specific RT primer if the melting temperature of a hybrid of the RNA template to the 5' 7-9 nucleotide sequence is about 5°C to about 2OºC lower than the melting temperature of the corresponding reverse PCR primer/DNA hybrid. In some embodiments of the invention, a reverse PCR primer may comprise, in addition to the sequence of an RT primer and the 3' located sequences discussed above, additional sequences, such as a universal sequence (e.g. from M 13), at the 5' end of the primer. In this embodiment, the Tm of the PCR primer/template hybrid can be up to about 35ºC higher than the Tm of a hybrid containing just the embedded RT primer.

The inventors have demonstrated the superiority in an RT-PCR method of using an 8-or 9 mer RT primer designed according to a method of the invention compared to using a comparable 12 to 14-mer RT primer designed on the basis of DNA/DNA hybridization. The Examples herein show experiments in which Real Time PCR was used to detect amplification of various genes. The delta Ct value for these genes crossed the threshold of detection at a lower value using an 8 or 9 mer reverse transcription primer than when using a 12 to 14-mer primer designed using DNA/DNA hybridization kinetics; this resulted in a significant increase in sensitivity using the 8 or 9 mer primers.

Target-specific RT primers and target-specific PCR primers for more than one target can be used in a single reaction. The temperature range used during the RT step (generally from about 37°C to about 42°C) allows the primers to hybridize to their specific RNA template in the reverse transcription reaction, but not during the PCR step. Thus, the primers of the invention can be used as a panel of primers while preserving the enhanced nature of signal detection of gene-specific priming and preventing primer-dimer formation. In such a multiplex cDNA synthesis reaction, it is preferable to ensure against primer competition and secondary priming by comparing and selecting sequences that do not share significant regions of homology. Conventional software programs can be used to simplify analyzing reactions and insuring optimum length primers. A combination of primers, of various lengths determined by sequence or prior design, can be used together.

In embodiments of the invention, the RT-PCR method is applied to formalin fixed, paraffin-embedded tissue (PET) and/or to a degraded RNA target (e.g. an RNA target that is between about 60-200 nt in length, e.g. about 100- 200 nt in length). In embodiments of the invention, the RT-PCR method is a method of expression analysis; is a Quantitative Real-Time PCR method; and/or is used for forensic or diagnostic analysis. In one embodiment, a target- specific nucleic acid product (e.g. , DNA product) of an RT-PCR method of the invention is further hybridized to an array of probes.

One aspect of the invention is a method for detecting expression of a marker of interest in a cell or tissue, comprising reverse transcribing RNA from the cell or tissue by a method of the invention to produce a marker-specific DNA product; and amplifying the marker-specific DNA product. In one embodiment, the method comprises (a) designing an optimized nucleic acid RT primer by a method of the invention; (b) reverse transcribing the RNA, using the optimized target-specific primer, under conditions effective for specific hybridization, to generate a target- specific DNA product; and (c) amplifying the target-specific DNA product, e.g., using PCR, to generate an amplified product The presence of an amplification product indicates expression of a marker in the cell or tissue. Multiple markers may be detected simultaneously, using appropriate RT and PCR primers specific for each of the RNAs to be detected.

A "marker," as used herein, refers to a sequence (e.g. a gene or gene product) that is expressed by a cell or tissue of interest, at least to a degree which is detectable by a method of the invention, but is expressed to a much more limited degree (e.g., below the limit of detection by a method of the invention) by other cells or tissues that are tested. In one embodiment, the marker is expressed at a level at least two times, e.g. at least about 5 times, 10 times or more, mat of control sequences from cells or tissues other than the cell or tissue of interest.. A marker can reflect such specific expression of a cell or tissue of interest, or it can reflect the presence of a disease (e.g. cancer, or a particular type of cancer).

In embodiments of this method, the RNA is an mRNA; the primer is DNA, LNA or RNA; the primer is DNA and the kinetics of hybridization are optimized for a hybridization temperature of about 37ºC to about 42ºC; and/or the optimized target-specific primer is about 7-9 nt in length (e.g. 8-9 nt in length)..

In a preferred embodiment, the detection of the expression of the marker comprises quantitating the amount of expression, compared to a baseline value. A "baseline value," as used herein, represents a control value. Examples of suitable baseline controls will be evident to the skilled worker. For example, the baseline value in an assay for expression of a cancer marker may be obtained by detecting the amount of expression of the cancer marker in a sample from a normal cell or tissue, or in a "pool" of such normal samples. The pooled values may be available in a database compiled from such values. Other controls that can be used include suitable internal controls, such as positive controls, negative controls, and quantitation controls, examples of which will be evident to the skilled worker. Suitable controls include markers that are constitutively expressed, such as housekeeping genes, including S 15, beta-actin, beta 2 microglobulin, GAPDH, HPRT, PGK and ALAS (human 5-aminoleculinate synthase).

In the assays described herein, a given polynucleotide may or may not be expressed in an increased or decreased amount in a sample compared to a baseline value. In a general sense, this invention relates to methods to determine if a gene product is expressed in an increased or decreased amount, irrespective of whether such increased or decreased expression is detected.

The degree of expression of a marker can be measured by detecting and/or quantitating target-specific nucleic acid products or, preferably, amplified products thereof. Such methods are conventional. For example, the products may be labeled with one or more labeling moieties. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, such as ³²P, ³³P or ³⁵S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like. In one embodiment, a fluorescent dye is incorporated directly by using a fluorochrome conjugated nucleotide triphosphate (e.g. Cy3-dUTP) or through a secondary coupling reaction by first incorporating an amino allyl conjugated nucleotide triphosphate (e.g. amino allyl-dUTP) followed by chemical coupling of the fluorochrome (e.g. NHS-Cy3).

Exemplary dyes include quinoline dyes, triarylmethane dyes, phthaleins, azo dyes, cyanine dyes and the like. Preferably, fluorescent markers absorb light above about 300 nm, preferably above 400 nm, and usually emit light at wavelengths at least greater than 10 nm above the wavelength of the light absorbed. Specific preferred fluorescent maikeis include fluorescein, pliycoerythim, rhodamine, hssamme, and Cy3 and Cy5 available fiom Amersham Pharmacia Biotech (Piscataway, N.J.).

Labeling can be earned out diumg an amplification reaction, such as PCR, or by nick translation or 5' or 3'-end-labelmg ieactions In one embodiment, the labeling moiety is incorporated after the PCR ieaction has been completed For example, biotm can fust be incorporated duπng a PCR reaction. Unbound polynucleotides are iinsed away so that the only biotm iemaming is that which was mcorpoiated duimg the PCR reaction Then, an avidm-conjugated fluoiophoie, such as avidin-phycoerythim, that binds with high affinity to biotm is added In another embodiment, the labeling moiety is incorporated by intercalation into the (double stianded) PCR pioduct In this case, an intercalating dye such as a psoralen-linked dye can be employed

In another embodiment, Quantitative Real-Time PCR, which is a 5¹ fluorogenic nuclease assay for determining abundance of mRNA species, is used The basis of this system is to continuously measure PCR pioduct accumulation A variety of suitable methods of detection will be evident to the skilled worker For example, SYBR Gieen, Amplifluours, molecular beacons 01 Scorpions can be used In a preferred embodiment, a fluorogenic oligonucleotide probe called a TaqMan™ probe is used This piobe is composed of a short (~20-25 bases) oligodeoxynucleotide containing, at its 5' and 3' ends, a fluorescent dye and a quencher moiety The oligonucleotide probe sequence is homologous to an internal target sequence present m the PCR amplicon When the probe is intact, emission from the fluoiescent dye is quenched by the quencher Duπng the extension phase of PCR, the probe is cleaved by 5' nuclease activity of Taq polymeiase, theieby ieleasmg the repoiter from the oligonucleotide quencher and producing an increase in reporter emission intensity This increase in fluorescence is detected and plotted versus PCR cycle number to produce a continuous measure of PCR amplification See the Examples herein for illustiations of how this method can be used

The RT-PCR methods, including the detection methods, of the invention can be applied to samples from any of a variety of cells or tissues, in any of a variety of forms The cell may be an isolated cell, or it may be part of a tissue The cell or tissue may be preserved or non-preserved Fixed (e g with formalin), embedded tissue, fresh tissue, and fresh-frozen tissue and body fluids may be used

An advantage of the present pπmeis and RT-PCR methods is that they can be used to identify specific nucleic acids in a cell or tissue that has low levels of RNA or DNA Tissues with low levels of nucleic acids include body fluids (e g , peripheral blood, urine, cerebrospinal fluid, pulmonary lavage, gastric lavage, bile, vaginal secretions, seminal fluid, aqueous humor, vitreous humor, etc ) Another advantage of the present pπmers and RT-PCR methods is that they can be used to identify specific nucleic acids m tissues m which the nucleic acids are highly degraded oi otherwise modified fiom their native state For example, RNA which is degraded to fragments that are about 60 to about 200 nucleotides in length, e g about 100 to about 200 nt m length, can be detected by methods of the invention Examples of these tissues include fixed (e g , formalm-fϊxed) tissues, and embedded (e g , paraffin- embedded) tissues Such tissues are widely collected from biopsies and other procedures and remain available because they are storage stable at room tempeiature Other compromised tissues that can be characterized by methods of the invention include tissue samples obtained from an autopsy or from a crime scene

Methods of the invention can be used to detect any of a variety of nucleic acid targets, e g, RNAs from genes whose expiession patterns are of interest Many examples of suitable taigets will be evident to the skilled woiker Suitable nucleic acids targets include nucleic acids (e.g., DNA or KNA) from the genomes of, or expressed by, infectious agents (e.g., pathogens); or host gene products whose expression is stimulated or inhibited in response to infection by the agents. These infectious agents include, for example, HIV, retroviruses, cytomegalovirus, adenovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Herpes I virus, Herpes II virus, influenza virus, polio virus, papilloma virus, vaccinia and smallpox virus; non-viral pathogens, including bacteria, protozoan parasites, etc. Among the many types of sequences of pathogenic organisms that can be detected by methods of the invention are sequences encoding rRNAs, reverse transcriptases and proteases. One of skill will recognize a variety of additional infectious agents whose presence can be detected with the present primers and methods. One of skill in the art can readily design and optimize suitable RT and PCR primers to detect an infectious agent of interest.

In other embodiments, primers and methods of the invention are used for diagnostic or prognostic methods, employing gene expression patterns in cells or tissues of interest. For example, autopsy samples, or samples from dried blood, biological fluid, vaginal secretions, epithelial cells or the like can be used. In another embodiment, the primers and methods are used for forensic analysis. For example, samples of unknown origin taken from a crime scene can be used in the reconstruction of the crime scene. A skilled worker will recognize a variety of tissue-specific markers that can be used to identify the source of a sample. Some of the genes whose analysis is particularly useful for forensic analysis are listed in Table 1:

Table 1

MMP gene family (1 thru 11)

PR (progesterone receptor)

SP-Al and SP-A2 (Surfactant apoprotein)

IL-lalpha,IL-beta,IL-6

TNF alpha beta-actin, GAPDH, CK 19, S 15 (housekeeping genes) cyclophilin beta-spectrin protamine 1 mucin 4

18S rRNA statherin histatin 3

PRB1, PRB2, PRB3

In one embodiment, RT primers and methods of the present invention are used to detect transcripts of cancer genes. Representative cancer genes that can be detected by the methods of the invention include, but are not limited to, cancer genes related to breast, esophagus, lung, colon, skin, brain, bone, salivary gland, liver, stomach, pancreas, gall bladder, kidney, bladder, prostate, lymphoma, leukemia and sarcoma. The cancer genes can be detected in a primary cancerous tumor or in a secondary (metastatic) cancerous tumor.

Many genes have been identified that can be used to detect various types of cancers in diagnostic assays. To detect breast cancer, for example, RT primers directed to the following genes whose expression has been correlated with breast cancer genes can be used: β2m (beta macroglobulin), mam (mammaglobin), PIP (prolactin-inducuble protein), KSl/4 (epithelial cell adhesion molecule), PSE (prostate-specific Ets factor), BRCAl or 2, and/or CEA (carcinoembryonic antigen). To detect lung and colon cancer, RT primers directed to genes including β₂n, CEA,

CKl 9 (cytokeratin), muc1 (mucin 1), and/or lunx (lung-specific X protein) can be used. For esophageal cancer, primers directed to genes including β2m, ErbB2, EpCam, PDEF, HoxC6 and POTE can be used. Other cancer - related genes that can be used are well-known to those of skill in the art. These genes include, e.g., EGF-R, HER- 2/neu; FGF-R4; P21/waf/Cipl/; MDM2; SBEM, XA G and TFF. Other suitable cancer markers are included in Table 2 below, which also lists non-cancer markers that can be detected by methods of the invention. Panels of primers for detecting cancer genes, such as the genes listed below in Table 2, can be used.

Table 2

In one embodiment of the invention, the cancer genes which are detected do not include one or more of the following genes: PIP, CK19, CEA, PSE, β2m, mam, mucl, SBEM, ErbM2, EpCam, PDEF, HoxC6, POTE, XAG, or TFFl. In another embodiment, one or more of c-myc, PIP and keratin- 19 are not used to detect cancer, e.g. metastatic breast cancer.

[THIS SPACE INTENTIONALLY LEFT BLANK]

Other suitable cancer markers are included in the following Table 3.

Abbreviation Cancer

AEL acute eosinophilic leukemia

AL acute leukemia

ALCL anaplastic large cell lymphoma

ALL acute lymphocytic leukemia

ALL acute lymphocytic leukemia

AML acute myelogenous leukemia

APL acute promyelocytic leukemia

B-CLL B-cell Lymphocytic leukemia

B-NHL B-cell Non-Hodkin Lymphoma

CLL chronic lymphatic leukemia

CML chronic myeloid leukemia

CMML chronic myelomonocytic leukemia

CNS central nervous system

DFSP dermatofibrosarcoma protuberans

DLBL diffuse large B-cell lymphoma

DLCL diffuse large-cell lymphoma

GIST gastrointestinal stromal tumour

JMML juvenile myelomonocytic leukemia mucosa-associated lymphoid tissue

MALT lymphoma

MDS myelodysplastic syndrome mediastinal large cell lymphoma with

MLCLS sclerosis

MM multiple myeloma

MPD Myeloproliferative disorder

NHL non Hodgkin lymphoma

NSCLC non small cell lung cancer

T-ALL T-cell acute lymphoblastic leukemia

TGCT testicular germ cell tumour

T-PLL T cell prolymphocytic leukaemia.

One of skill in the art can readily design target-specific RT and forward and reverse PCR primers to detect a gene of interest, such as the cancer markers indicated herein. Methods for designing RT primers are described elsewhere herein. Parameters for designing PCR primers are well-known in the art. For example, it is often desirable to select a forward PCR primer which spans two adjacent introns, so as to reduce background amplification of potential contaminating genomic DNA. For further guidance in designing and using PCR primers, see discussions in Innis et ah, PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc. Harcourt Brace Jovanovich, Publishers,, current edition; or Dieffenbach et al., supra.

Table 4 lists exemplary primer sets (RT primers, forward and reverse PCR primers) for five breast cancer- associated genes and a housekeeping gene; the bolded sequences indicate suitable 8- or 9-mer gene-specific RT primers corresponding to the reverse PCR primers. For /32m, RT primers of 8 or 9 nt are indicated. When an 8-mer RT primer is indicated for a gene {e.g. for PSE), a suitable 9-mer would contain an additional nucleotide at the 3' end of the indicated RT primer. That is, for PSE, the 9-mer would be AGCCACTTC. When a 9-mer RT primer is indicated for a gene, the corresponding 8-mer would consist of the 5' most 8 nts {e.g., for MAM, the corresponding 8-mer would be CTGCAGTT).

Table 4

Gene Sequence AE Intron size

(bp)

Im F 5' -GCCGTGTGAACCATGTGACTTT (SEQ ID NO: 1) 0.9 626 R 5' -CCAAATGCGGCATCTTCAAA (SEQ ID N0:2) 1246 R 5' -CCAAATGCGGCATCTTCAAA (SEQ ID N0:3) 1246

MAM F 5' -CGGATGAAACTCTGAGCAATGT (SEQ ID N0:4) 1.0 1887 R 5' -CTGCAGTTCTGTGAGCCAAAG (SEQ ID N0:5)

PIP F 5' -GCCAACAAAGCTCAGGACAAC (SEQ ID N0:6) 1.0 2974 R 5' -GCAGTGACTTCGTCATTTGGAC (SEQ ID N0:7)

KS1/4 F 5' -CGCAGCTCAGGAAGAATGTG (SEQ ID N0:8) 1.0 3879 R 5' -TGAAGTACACTGGCATTGACGA (SEQ ID N0:9)

PSE F 5' -AGTGCTCAAGGACATCGAGACG (SEQ ID NO:10) 1.0 2835 R 5' -AGCCACTTCTGCACATTGCTG (SEQ ID NO: 11)

CEA F 5' -GGGCCACTGTCGCATCATGATTGG (SEQ ID 1.0 1831 NO: 12)

R 5' -TGTAGCTGTTGCAAATGCTTTAAGAAGAAGC (SEQ ID NO: 13)

A skilled worker can readily determine comparable sets of primers for any gene of interest.

The present invention provides pairs of PCR forward and reverse PCR primers which are matched with RT primers, for detecting the expression of any gene of interest. The primers can be packaged kits suitable for detecting the expression of the genes. In one embodiment of the invention, target-specific nucleic acid products (e.g. DNA products) of the invention or, preferably, amplified products thereof, are analyzed by hybridizing them to a collection of probes representing markers of interest. For example, products reflecting the expression of cancer markers can be analyzed by hybridizing them to an array, such as a microarray, of probes for cancer markers of interest on a gene chip. In one embodiment, nucleic acid products that have been generated from an RNA sample by using multiple sets of RT and PCR primers are identified individually by virtue of their hybridization to probes at discrete sites on a gene chip. The assay can be a high throughput assay. For example, from a single PET sample, an investigator can determine the amount of expression of any of a variety of cancer markers, as well as the amount of expression of positive, negative and/or quantitation controls.

The nucleic acid primers, target-specific DNA products, or amplified products thereof, may take any of a variety of forms. For example, at least one phosphate, sugar and/or base moiety in the helix may be modified. In some embodiments, for example, a phosphate may be modified as a phosphorothioate, a phosphoridothioate, a phosphoramidothioate, a phosphoramidate, a phosphordiimidate, a methylphosphonate, an alkyl phosphotriester, 3 '- aminopropyl, a formacetal, or an analogue thereof.

The nucleic acids can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups. Suitable modified base moieties include, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-ω-thiouiidine, 5-carboxymethyl- aminomethyl uracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isoρentenyladenine, 1-methylguanine, 3- methyl-cytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino- methyl-2-thiouracil, β-D-mannosylqueosine, 5-methoxy-carboxymethyluracil, 5-methoxyuracil-2-methylthio-N6- iso-pentenyladenine, uracil-5-oxyacetic acid, butoxosine, pseudouracil, queuosine, 2-thio-cytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-t-oxyacetic acid, 5- methyl-2-thiouracil, 3(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine.

The nucleic acid may comprise at least one modified sugar moiety including, but not limited, to arabinose, 2-fluoroarabinose, xylulose, and hexose.

The nucleic acid may comprise a modified phosphate backbone synthesized from one or more nucleotides having, for example, one of the following structures: a phosphorothioate, a phosphoridothioate, a phosphoramidothioate, a phosphoramidate, a phosphordiimidate, a methylphosphonate, an alkyl phosphotriester, 3 '- aminopropyl and a formacetal or analog thereof.

The nucleic acid may be an α-anomeric oligonucleotide which forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al. (1987), Nucl. Acids Res. 15:6625-6641).

The nucleic acid may be conjugated to another molecule, e.g., a peptide, a hybridization-triggered cross- linking agent, a hybridization- triggered cleavage agent, etc., all of which are well-known in the art.

Methods of making and isolating (e.g. purifying) nucleic acids of the invention are conventional. Nucleic acid primers of the invention may be synthesized, in whole or in part, by standard synthetic methods known in the art. See, e.g., Caruthers et al. (1980) Nucleic. Acids Symp. Ser. (2) 215-233; Stein et al. (1998) , Niicl. Acids Res. 16, 3209; and Sarin et al. (1988), Proc. Natl. Acad. Sci. U.S.A 85, 7448-7451. An automated synthesizer (such as those commercially available from Biosearch or Applied Biosystems) may be used. cDNA primers can be cloned and isolated by conventional methods; can be isolated from pre-existing clones, or can be prepared by a combination of conventional synthetic methods.

RNA to be reversed transcribed and amplified by methods of the invention can be isolated according to any of a number of methods well known to those of skill in the art. For guidance as to methods of purification of nucleic acids, as well as other molecular biology methods used in aspects of the invention, see e.g. Sambrook, et al. (1989), Molecular Cloning, a Laboratory Manual, Cold Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al. (1995). Current Protocols in Molecular Biology, N. Y., John Wiley & Sons; Davis et al. (1986), Basic Methods in Molecular Biology, Elsevier Sciences Publishing,, Inc., New York; Hames et al. (1985), Nucleic Acid Hybridization, IL Press; Dracopoli et al. Current Protocols in Human Genetics, John Wiley & Sons, Inc.; and Coligan et al. Current Protocols in Protein Science, John Wiley & Sons, Inc.

For example, total RNA can be isolated by procedures including guanidium/phenol/chloroform extraction or the TRJZOL total RNA isolation reagent (Life Technologies, Gaithersburg, Md.); and mRNA can be isolated using oligo d(T) column chromatography or glass beads. Methods for isolating RNA from PET samples are conventional. See, e.g., Korbler et al. (2003) Experimental and Molecular Parasitology IA_., 336-340.; Coombs et al. (1999) Nucleic Acids Research 27, el2; Specht et al. (2001) American Journal of Pathology 158, 419- 429;Mikhitarian et al. (2004) BioTechniques 36, 474-478; Masuda et al. (1999) Nucleic Acids Research 27, 4436- 4443 ;or Godfrey et al. (2000) Journal of Molecular Diagnostics 2, 84.

Another aspect of the invention is a set of a limited number of RT primers of the invention. For example, the set may comprise RT primers specific for a limited number of cancer genes (markers), e.g. fewer than about 50, about 100, or about 150 markers, or more. Optionally, each of these genes may be represented by multiple primers (e.g. about five or fewer primers) corresponding to different sequences along the length of the gene. In embodiments of the invention, the RT primers are selected from the RT primers listed in Table 4; and/or they are other primers corresponding to those cancer markers; and/or they are primers for other cancer markers. Optionally, the set may also include RT primers specific for genes for positive, negative and/or quantitative controls (e.g. housekeeping genes). In general, a set of RT primers of the invention consists of fewer than about 200 RT primers. The RT primers in a set may be primers that are optimized by methods of the invention; primers that are about 7-9 nt in length (e.g., primers that are 8-9 nt in length); and/or combinations thereof. It is noted that a set of RT primers of the invention differs from sets of random primers (e.g. random 6-mer or 9-mer primers), at least because in the inventive primer sets, only a small, selected subset of primers is present.

Another aspect of the invention is a kit suitable for performing one of the methods of the invention. One embodiment is a kit for detecting the presence and/or amount in a sample from a subject of RNA transcribed from one or more markers of interest. The kit may comprise a set of RT primers of the invention, as discussed above. In embodiments of the invention, the kit may further comprise, for one or more of the RT primers, a target-specific reverse PCR primer and, optionally, a target-specific forward PCR primer, wherein the reverse PCR primer comprises the RT primer. In embodiments of the invention, the reverse PCR primer comprises the RT primer on its 5' end, and further comprises at least about 8 additional target-specific nucleotides on its 3' end. The kit may comprise target-specific RT and PCR primers for more than one marker. In one embodiment, the kit comprises one or more RT primers selected from the RT primers listed in Table 4; optionally, one or more corresponding reverse PCR primers such as those listed in Table 4; and, optionally, the corresponding forward PCR primers. Alternatively, a kit of the invention may comprise comparable RT primers and, optionally, and forward and/or reverse PCR primers, corresponding to any of the genes discussed herein (including controls, such as housekeeping genes)..

A kit of the invention may further comprise means for carrying out reverse transcription and/or PCR amplification. The kit may comprise one or more reagents that facilitate reverse transcription and/or PCR amplification, and/or detection of the nucleic products of the RT and/or PCR reactions.

A kit of the invention may comprise instructions for performing a method, such as a diagnostic method. Other optional elements of a kit of the invention include suitable buffers, media or buffer components, or the like; a computer or computer-readable medium for storing and/or evaluating the assay results; containers; or packaging materials. Reagents for performing suitable controls may be included. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in single reaction form for diagnostic use.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

EXAMPLES

Example 1 - Materials and Methods

DNA RT primers (8-9 nucleotides in length) were designed for seven cancer-markers and a housekeeping gene: β2-microglobin (β2m), mammaglobin (mam), prolactin-inducible protein (PIP), epithelial cell adhesion molecule (KS 1/4), prostate-specific antigen (PSE), carcinoembryonic antigen (CEA), mucin 1 (MUC-I) and lung- specific X protein (LUNX). The RT primers were designed on the basis of, among other factors, the temperature of the hybridization reaction between the RT primer and the RNA template, and the T_m of the RNA/DNA hybrid. For each gene, the RT primers correspond to the 5 '-end of a reverse primer used for PCR of the gene. The RT and PCR primers are shown in Table 4. The annealing temperatures for the 8 to 9-mer RT primers are 37° to 42ºC, a range that allows the primers to hybridize to their specific template in the reverse transcription reaction, but not during the PCR.

Comparison primers (12-14 nt in length) were also prepared for each of the above-mentioned genes. These primers were designed by conventional methods, taking into account the Tm of DNA/DNA hybrids (i.e. calculating the Tm of a hybrid between a primer made of DNA and a DNA template).

A summary of the experimental and comparison RT primers used in Examples II and III is shown in Table 5. Table 5

RNA from 40 micron sections of formalin-fixed, paraffin embedded breast cancer tissues was isolated following the method of Specht et al. (2001) Am J Pathol.158, 419-29, with some modifications. PET sections were deparaffmized twice with 1 ml of xylene at 37ºC for 20 min. The pellet was washed with 0.5 ml of 100%, 90%, then 70% ethanol and air dried at room temperature. The dried pellet was resuspended in 200 μl of 500 mg/ml proteinase K, 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 2% SDS, ρH7.3 and incubated at 60ºC for 16 hours. RNA was extracted, using an equal volume, withphenol:chloroform:isoamyl alcohol (25:24:1) and the non-organic aqueous layer transferred to a clean, RNAse-free tube. The RNA was precipitated using 0.1 volume 3 M sodium acetate, 10 μg glycogen as carrier, 1 volume isopropanol and an incubation at -20ºC overnight. After a 30 min centrifugation at 12,000 rpm, the RNA pellet was washed with 70% ethanol, and allowed to air dry. The sample was resuspended in 10 μl Rnase-free water and stored at -20ºC. RNA was quantified by spectrophotometry at 260 nm.

The mRNA was reverse transcribed as follows: cDNA was made from 5 μg of total RNA using 500 ng of the panel of RT gene-specific primers shown in Table 5. RNA was reverse- transcribed with 200 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA) in a reaction volume of 20 μl (10 min at 70ºC, 50 min at 42ºC, 15 min at 70ºC).

Real-time RT-PCR analyses were performed on a 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The standard reaction volume was 10 μl and contained Ix QuantiTect^® SYBR^® Green PCR Master Mix (Qiagen, Valencia, CA, USA), 0.1 U AmpErase^® UNG enzyme (Applied Biosystems, Foster City, CA, USA), 0.7 μ1 cDNA template, and 0.25 μM of both forward and reverse primer. All primers were designed to be intron-spanning to preclude genomic DNA amplification. β2m was used as an internal control. The initial step of PCR was 2 min at 50ºC for AmpErase^® UNG activation, followed by a 15 min hold at 95ºC. Cycles (n=40) consisted of a 15-s denaturation step at 95ºC, followed by 1 min annealing/extension step at 60ºC. The final step was a 1-min incubation at 60ºC. All reactions were performed in triplicate. Real-time RT- PCR data were quantified in terms of cycle threshold (Q) values. Q values are inversely related to the amount of starting template: high Q values correlate with low levels of gene expression, whereas low C_t values correlate with high levels of gene expression. The specificity of the PCR products was confirmed by dissociation profile analyses. The fold difference in signal detection was calculated using the formula (l+AE)^ΛCt, where AE is the amplification efficiency and ΔCt = Ct _{12-14 mer} gene-specific primers ^{- Ct} 7-9 mer gene-specific primers-

Example II - RT-PCR using 8-9 nt RT primers of the invention is at least as efficient as RT-PCR using corresponding 12-14 mer RT primers

Using the methods discussed in Example I, two sets of reverse transcription reactions were carried out. In the first set, 5 μg of total RNA was converted to cDNA, using 500 ng of a panel of truncated 12-14 nt gene-specific primers [β2-microglobin (β2m), mammaglobin (mam), prolactin-inducible protein (PIP), epithelial cell adhesion molecule (KS 1/4), prostate-specific antigen (PSE), and carcinoembryonic antigen (CEA)]. The second set was carried out in the same manner, except 8-9 nt gene-specific primers were used.

Real-time RT-PCR analysis for individual markers was carried out as indicated; the results are summarized in Table 6.

Table 6

In Table 6, RT-PCR data from a number of different tissue samples are summarized (indicated as sample 274, 1040, 376, etc.) "Delta Ct" and "fold difference" are defined in Example I. Note in particular the column labeled "Fold Diff." This column summarizes the differences between Ct values obtained with the 12-14 mer primers compared to the results obtained with the 8-9 mers.

The data show that the panel of thermodynamically defined (8-9-mer) truncated gene-specific RT primers used during reverse transcription provided superior results (a significant enhancement of gene fragment detection from the PET samples containing highly degraded RNA) compared to 12-14 mer gene-specific RT primers.

Example III - When RT-PCR is carried out using a mixture comprising an 8-9 nt RT primer for one of the genes being amplified and 12-14 mer RT primers for several other genes, the improved amplification of the gene using the 8-9 mer primer is not negatively affected by the presence of the longer primers in the reaction mixture.

Using the methods discussed in Example I, two sets of reverse transcription reactions were carried out. In the first set, 5 μg of total RNA was converted to cDNA, using 500 ng of a panel of 12-14 nt gene-specific RT primers as described in Example II. A second set of reactions was carried out in the same way, except the RT primers were a mixed primer set: the RT primer for /32M was 8 nt, and the RT primers for the other genes were 12- 14 mers. A third set of reactions was also carried out, using a slightly different mixture of RT primers: the RT primer for /32M was 9 nt, and the RT primers for the other genes were 12-14 mers. The sequence of the three different β 2m primers (12, 9 and 8 nt in length) is shown in Table 6.

The real-time PCR data for β2m from the cDNA generated by the 12-14 mer primer set were compared to the data for β2m from the cDNA generated by the two "mixed" primer sets. As shown in Table 7, the Ct value for amplification with the β2m primer when all of the primers in the reaction mix were 12-14 mers was 29.41. The value for the 9-mer β2m primer in the presence of the 12-mer primers was lower (26.97); and the value for the 8- mer β2m primer in the presence of the 12-mers was even lower (24.28). As indicated in the Table, the calculated fold difference compared to the 12-mer was 5-fold for the 9-mer, and 27 fold for the 8-mer. That is, the 8 and 9-mer primers gave superior results compared to the 12-mer primer; and the superior results were not negatively influenced by the presence in the reaction mix of 12-14 mer primers corresponding to other genes.

Table 7

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications (including U.S. provisional patent application Ser. No. 60/691,247, filed June 17, 2005), patents, and publications cited above and in the figures, are hereby incorporated in their entirety by reference.

Claims

WE CLAIM

1. A method for designing an optimized nucleic acid primer for reverse transcription of an RNA target of interest, comprising selecting a region of the RNA target for designing a target-specific primer, and optimizing the kinetics of hybridization of candidate target-specific primers corresponding to that region of the RNA, thereby designing an optimized target-specific RT primer.

2. The method of claim 1, wherein the RNA is an mRNA.

3. The method of claim 1, wherein the primer is DNA.

4. The method of claim 1, wherein the primer is linked nucleic acid (LNA) or RNA.

5. The method of claim 1, wherein the primer is DNA, and the kinetics of hybridization are optimized for a hybridization temperature of about 37ºC - about 42ºC.

6. The method of claim 5, wherein the optimized target-specific primer is about 8-9 nucleotides in length.

7. The method of claim 5, wherein the optimized target-specific primer is 7-9 nucleotides in length.

8. A method for reverse transcribing one or more RNA targets of interest, in a reaction mixture, comprising hybridizing target-specific RT primers designed by the method of claim 1, and reverse transcribing the RNA(s) to generate target-specific DNA product(s), wherein the number of RT primers in the mixture is no more than five times greater than the number of RNA targets.

9. The method of claim 8, wherein

(i) the RNA is an mRNA;

(ii) the primer is DNA, LNA or RNA;

(iii) the primer is DNA and the kinetics of hybridization are optimized for a hybridization temperature of about 37ºC - about 42ºC; and/or (iv) the optimized target-specific primer is about 7-9 nucleotides in length or is 8-9 nucleotides in length.

10. A method for reverse transcribing one or more RNA targets of interest, in a reaction mixture, comprising hybridizing target-specific RT primers which are 8-9 nucleotides in length to the RNA(s), and reverse transcribing the RNA(s) to generate target-specific DNA product(s), wherein the number of RT primers in the mixture is no more than five times greater than the number of RNA targets.

11. An amplification method, comprising amplifying the target-specific DNA product(s) of claim 8 or claim 10.

12. The method of claim 11, which is an RT-PCR method.

13. The RT-PCR method of claim 12, wherein the PCR amplification comprises amplifying the DNA product using a target-specific forward PCR primer and a target-specific reverse PCR primer, wherein the reverse PCR primer comprises the RT primer.

14. The RT-PCR method of claim 13, wherein the reverse PCR primer comprises the RT primer on its 5' end, and further comprises at least about 8 additional target-specific nucleotides on its 3' end.

15. The RT-PCR method of claim 12, wherein the reverse transcription step utilizes an annealing temperature from about 37ºC to about 42ºC.

16. The RT-PCR method of claim 12, wherein the RNA is from formalin fixed, paraffin-embedded tissue.

17. The RT-PCR method of claim 12, wherein the method is applied to RNA targets that are about 60 to about 200 nucleotides in length.

18. The RT-PCR method of claim 12, wherein the reverse transcription step is carried out at about 37ºC to about 42ºC, the RT primer is 8-9 nucleotides in length, and the yield of amplified target is greater than in an RT-PCR method in which the transcription step is carried out at about 37ºC to about 42ºC and the RT primer is 12-14 nt in length.

19. The method of claim 12, further wherein the amplified DNA product is hybridized to an array of probes.

20. The method of claim 12, which is a method of expression analysis.

21. The method of claim 12, in which the PCR is Real-Time PCR.

22. The method of claim 12, which is a method of forensic analysis.

23. An optimized nucleic acid RT primer made by the method of any of claims 1-7.

24. A set of RT primers made by the method of claim 1, wherein the set consists of no more than about 200 RT primers.

25. A set of RT primers that are 8-9 nt in length, wherein the set consists of no more than about 200 RT primers.

26. A method for detecting expression of one or more markers of interest in a cell or tissue, comprising a) reverse transcribing RNA from the cell or tissue, in a reaction mixture, using one or more RT primers designed by the method of claim 1, to produce a marker-specific DNA product(s); wherein the number of RT primers in the reaction mixture is no more than about five times greater than the number of markers of interest; and b) amplifying the marker-specific DNA product(s) to generate an amplification produces), the presence of an amplification product indicating expression of the marker in the tissue.

27. The method of claim 26, wherein the amplifying in step (c) is PCR which comprises using a marker-specific forward PCR primer and a marker-specific reverse PCR primer, wherein the reverse PCR primer comprises the RT primer.

28. The method of claim 26, wherein

(i) the RNA is an mRNA;

(ii) the primer is DNA, PNA, LNA or RNA;

(iii) the primer is DNA and the kinetics of hybridization are optimized for a hybridization temperature of about 37ºC - about 42ºC; and/or (iv) the optimized target-specific primer is about 7-9 nucleotides in length or is 8-9 nucleotides in length.

29. The method of claim 26, wherein the RT primer is DNA, and the RT primer is optimized with regard to the kinetics of hybridization at a hybridization temperature of about 37ºC - about 42ºC.

30. The method of claim 26, wherein the RT primer is 8-9 nucleotides in length.

31. A method for detecting expression of a one or more markers of interest in a cell or tissue, comprising a) reverse transcribing RNA from the cell or tissue, in a reaction mixture, using an RT primer consisting of 8- 9 nucleotides that is specific for RNA transcribed from the marker, to produce a marker-specific DNA product; wherein the number of RT primers in the reaction mixture is no more than about five times greater than the number of markers of interest; and b) amplifying the marker-specific DNA product to generate an amplification product, the presence of an amplification product indicating expression of the marker in the tissue.

32. The method of claim 31, wherein the amplifying in step (b) comprises using a marker-specific forward PCR primer and a marker-specific reverse PCR primer, wherein the reverse PCR primer comprises the RT primer.

33. The method of claim 26 or claim 31, wherein the detection is quantitative.

34. The method of claim 26 or claim 31, wherein the tissue is a formalin-fixed, paraffin-embedded tissue.

35. The method of claim 31, wherein the RNA from the tissue is about 30-200 nucleotides in length.

36. The method of claim 26 or claim 31 , wherein the reverse transcription step is earned out at between about 37ºC and about 42ºC, and the yield of amplified target is greater than that when the target specific primer is 12-14 nt in length.

37. The method of claim 26 or claim 31, which is a forensic method.

38. The method of claim 26 or claim 31, wherein the marker is a virus-specific gene.

39. The method of claim 26 or claim 31, wherein the marker is a cancer gene.

40. The method of claim 39, wherein the cancer gene is related to breast, esophagus, lung, colon, skin, brain, bone, salivary gland, liver, stomach, pancreas, gall bladder, kidney, bladder, or prostate cancer, or is a lymphoma, leukemia or sarcoma.

41. The method of claim 40, wherein the cancer gene(s) is selected from the markers listed in Table 2 or Table 3.

42. The method of claim 26 or claim 31, wherein marker-specific RT primers and marker-specific PCR primers for more than one marker are used in a single reaction.

43. The method of claim 26 or claim 31 , further comprising comparing the amount of expression of the marker(s) of interest to a baseline value.

44. The method of claim 26 or claim 31, further comprising detecting the amount of expression of positive and/or negative control markers, and comparing the amount of expression of the marker(s) of interest to the amount of expression of the positive or negative control markers.

45. A kit for detecting one or more RNA targets of interest, comprising a set of RT primers of claim 24.

46. A kit for detecting one or more RNA targets of interest, comprising a set of RT primers of claim 25.

47. The kit of claim 45 or claim 46, further comprising, for each of the RT primers, a target-specific forward PCR primer and a target-specific reverse PCR primer, wherein the reverse PCR primer comprises the optimized RT primer.

48. The kit of claim 47, wherein the reverse PCR primer comprises the RT primer on its 5' end, and further comprises at least about 8 additional target-specific nucleotides on its 3' end.

49. The kit of claim 47, which comprises target-specific RT primers and target-specific PCR primers for more than five markers.

50. The kit of claim 45 or 46, wherein at least one of the RT primers is selected from the RT primers listed in Table 4.

51. The kit of claim 45 or 46, wherein at least one of the RT primers is specific for a housekeeping gene.

52. The kit of claim 51, wherein the housekeeping gene(s) is selected from the housekeeping genes listed in Table 2.

53. The kit of claim 45 or 46, which further comprises a. RT and/or PCR primers specific for positive, negative and/or quantitative control markers; and/or b. one or more reagents that facilitate reverse transcription of the RNA target and/or PCR amplification.

54. A method for generating a set of optimized nucleic acid RT primers corresponding to multiple sites on an mRNA expressed from a target of interest, comprising c. designing a first optimized nucleic acid RT primer from a first region of the RNA target, by the method of any of claims 1-7, d. designing a second optimized nucleic RT primer from a second region, about 100-300 away from the first primer, by the method of any of claims 1-7, and e. repeating the process until RT primers are designed corresponding to multiple sites on the RNA.

55. A method for reverse transcribing mRNA expressed from a target of interest, comprising hybridizing the set of optimized nucleic acid primers of claim 49 to the RNA, and reverse transcribing the RNA to generate a set of cDNAs to generate a set of target-specific DNA products.

56. The method of claim 55, wherein the expressed RNA is obtained from formalin-fixed, paraffin embedded tissue.

57. The method of claim 55, wherein the expressed RNA is fragmented into fragments that are about 60 to about 200 nucleotides in length.

58. An RT-PCR method, comprising PCR amplifying the target-specific DNA products of claim 49.

59. The method of claim 58, further comprising hybridizing the amplified target-specific DNA products to an array of probes.