JP2002512046A - Quantitative analysis of gene expression - Google Patents

Abstract

  (57) [Summary] A method is provided for determining the level of expression of a gene or gene panel of interest in a biological sample. The method involves generating a standard curve for each gene of interest by assaying a series of dilutions prepared from total RNA in a reverse transcriptase-5'-exonuclease PCR amplification assay. The reverse cycle-threshold cycle (Ct) for each amplified member is determined and plotted against the log of total RNA amount in a series of dilutions. This plot is used to determine the RNA equivalent at which the normalized RNA equivalent for the gene of interest in the sample is determined. The assay can be used to determine the effect of treating a sample on the expression level of a gene or panel of genes of interest.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0002] The present invention relates generally to quantitative reverse transcriptase-polymerase chain reaction ("PCR").
2) regarding the assay. More specifically, the present invention relates to a reverse transcriptase-PCR method using relative RNA standards to quantify target mRNA in a sample. The present invention has application in quantitative gene expression where determining changes in gene expression provides a measure of biological response and may be predictive or diagnostically useful.

BACKGROUND OF THE INVENTION [0002] Quantitative measurement of nucleic acid targets (DNA or mRNA) by PCR requires time-consuming post-amplification steps, which can lead to uncontrolled fluctuations and the risk of continued contamination. To address these issues, techniques have been developed that can more directly detect PCR products. Fluorescent energy transfer ("FET") PCR assays use non-extendable nucleic acid probes that are complementary to internal segments of target DNA. The probe is labeled with two fluorescent moieties that have the property that one emission spectrum overlaps the other excitation spectrum. As a result, the emission of the first fluorophore is largely quenched by the second fluorophore. The probe is present during PCR, and once the PCR product is created, the probe is susceptible to degradation via the native 5'-exonuclease activity of a DNA polymerase specific for the DNA hybridized to its complement. Nucleotide degradation of the probe allows the two fluorophores to separate in solution,
Decrease quenching and increase the intensity of emitted light. If the sample is measured on a fluorescent plate reader, no further post-amplification processing is required.

[0003] Typically, detection of DNA by quantitative PCR and mRNA by quantitative reverse transcriptase-polymerase chain reaction ("RT-PCR") requires the use of internal controls that are co-amplified with the target sequence. And As distinguished from the target sequence, the internal control can be a scrambled internal sequence, a variant of the amplicon, a deletion or insertion in the target amplicon, or a target primer sequence spliced on a heterologous DNA sequence. can do. However, differences in amplification efficiency between control and experimental nucleic acids can lead to significant differences in the amount of their PCR products. More recently, a quantitative PCR method has been developed that uses FET PCR to track the amount of PCR product that accumulates in real time, which determines the number of cycles required to reach a given amount of PCR product by the initial concentration of target nucleic acid. Used as a measure of However, these assays also require assumptions about co-amplified controls, and therefore relative amplification efficiencies in and between different samples, and controls during the logarithmic amplification phase of PCR. Moreover, it is time consuming and cumbersome to screen samples for expression levels of a large number of genes of interest to determine a "profile" of expression activity under normal conditions or stimuli. A major difficulty in the method of gene expression profiling has been the design of readily available techniques for quantitatively measuring the expression of a panel of genes with a wide variety of developmental levels.

SUMMARY OF THE INVENTION There is a need in the art for assays that allow the level of expression of a gene of interest to be determined without the problems inherent in conventional methods.
In addition, there is a need in the art for methods that allow one to easily test a gene or genes of interest for changes in expression upon stimulation.
Such a method should be applicable to small tissue samples, ultimately making direct comparisons between successive experimental samples from the same patient, potential background genetic variation,
As well as the analysis of clinical samples. The methods disclosed and claimed herein for measuring normalized RNA equivalents satisfy these criteria for gene expression profile measurements.

[0005] Accordingly, an object of the present invention is to provide a reverse transcriptase polymerase chain reaction ("RT-PCR
To provide a quantitative RNA rough measurement method based on a 5′-exonuclease assay for the above method. It is another object of the present invention to provide a method for determining a quantitative measure of gene expression of interest by measuring the relative level in a sample of target mRNA encoded by the gene of interest. Yet another object of the present invention is to provide a method for determining the effect of a stimulus on a quantitative measure of gene expression on the stimulus. Yet another object of the present invention is to provide a method for determining the expression level profile of a panel of genes in a sample. It is a further object of the present invention to provide a method for determining the effect of a stimulus on the profile of gene expression levels in a sample.

Thus, in one embodiment, a method is provided for determining a quantitative measure of the expression of a gene of interest in a biological sample by measuring the normalized RNA equivalent of the gene of interest in the sample. You. The method first involves generating a standard curve for the gene of interest using reverse transcriptase-5'-exonuclease polymerase chain reaction (RT-PCR). The standard curve was prepared by (i) preparing a series of reverse transcript standard dilutions from a total RNA extract with a known total RNA concentration, and (ii) transferring each member of the series of reverse transcript standard dilutions to 5′-exo. Amplify using a nuclease PCR assay, where the components of the assay include a forward primer, a reverse primer, a hybridization probe and a thermostable DNA polymerase, and the primers and probes bind to the gene of interest or its complement. Encoded mRNA
Specifically hybridize to a segment of the reverse transcript to form a stable hybrid duplex, the primer can prime the extension reaction, and the DNA polymerase can catalyze the primer extension reaction. Monitoring the fluorescence intensity from the 5'-exonuclease PCR assay in real time during each cycle of PCR for each member of a standard dilution of a series of reverse transcripts, having exonuclease activity; (Iv) Determine the threshold cycle (Ct) for each member of the standard dilution of the series of reverse transcripts, and (v) Ct versus the log of the total RNA amount for each member of the standard dilution of the series of reverse transcripts. From the plot of
Determined by a method comprising determining the NA equivalent and (vi) normalizing the RNA equivalent to provide a normalized RNA equivalent standard curve for the gene of interest. The normalized RNA equivalent for the gene of interest is determined by assaying the sample in RT-PCR and comparing the results therefrom to a standard curve.

In another embodiment of the invention, a method is provided for determining the effect of a treatment on a quantitative measurement of the expression of a gene of interest in a sample. This method uses the above-mentioned RT-PC
Creating a standard curve for the gene of interest using the R assay. Normalized RN of the gene of interest in the first untreated sample and the second treated sample
A equivalent is determined by assaying the first and second samples by RT-PCR and comparing the results obtained therewith to a standard curve. In yet another embodiment of the invention, a method is provided for determining a quantitative measure of the expression of a panel of genes of interest in a sample. The method involves generating a standard curve for each gene of interest using the RT-PCR assay described above and another different set of forward primers, reverse primers and hybridization probes for each gene of interest. including. The normalized RNA equivalent for the gene of interest in the sample is determined by testing the sample in RT-PCR for each gene of interest and taking the results obtained therefrom for each corresponding gene of interest. Determined by comparison to a standard curve.

It is a further object of the present invention to provide a method for determining the effect of a treatment on the quantitative measurement of expression of a panel of genes of interest in a first untreated sample and a second treated sample. It is. This method involves generating a standard curve for each gene of interest using the RT-PCR assay described above. The normalized RNA equivalent for each gene of interest in the first and second samples was determined by RT-PC
Determined by testing at R and comparing the results obtained therewith to the standard curve for each corresponding gene of interest. In a further embodiment, the present invention relates to a kit for performing the above method,
This includes (a) a forward primer, a reverse primer, and a hybridization probe, wherein the primer and probe specifically hybridize to the reverse transcript of the target mRNA molecule or its complement, and bind to its segment. A stable hybrid duplex can be formed, the primer can prime the extension reaction, (b) reverse transcriptase, (c) thermostable DNA polymerase, (d) instructions for performing the method. And, optionally, (e) other reagents necessary to perform the reverse transcription and / or PCR amplification reaction. These and other objects, embodiments and features of the present invention will become apparent to those of ordinary skill in the art upon reading the following disclosure and description of the invention.

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention will be described in molecular biology, microbiology, recombinant DN unless otherwise indicated.
Using conventional techniques of A-technology, cardiovascular physiology, and pharmacology, they are within the skill of those in the art. Such techniques are explained fully in the literature. For example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual,
Second Edition (1989); DNA Cloning, Vols. I and II (DN Glover ed. 1985)
); Perbal, B., A Practical Guide to Molecular Cloning (1984);
thods In Enzymology (ed. by S. Colowick and N. Kaplan, Academic Press, Inc.);
Transcription and Translation (Hames et al., Eds. 1984); Gene Transfer Vectors
For Mammalian Cells (JH Miller et al., (1987) Cold Spring Harbor Laborat
ory, Cold Spring Harbor, NY); Scopes, Protein Purification: Principle
s and Practice (2nd ed., Springer-Verlag); and PCR: A Practical approach
(McPherson et al. (1991) IRL Press). All patents, patent applications, and publications cited herein, whether listed above or below, are hereby incorporated by reference in their entirety. As used in this specification and the appended claims, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise. That is, for example, reference to "primer" includes two or more of its primers, reference to "probe" includes one or more of its probe, and reference to "gene of interest" to two or more. A gene, where "processing" includes one or more of its processing, and so forth.

A. Definitions In describing the present invention, the following terms will be used, and are intended to be defined as follows. The use of the term "standard curve" refers to the purpose of quantifying the amount of target RNA in a sample, where a known amount of RNA, preferably a series of dilutions, is assayed and the result obtained is an unknown amount of target RNA. RT- compared to the results obtained with the sample containing RNA
It is intended to mean a mathematical variant of the data obtained from the graphical description of the results of the PCR assay. A standard curve is generated for each gene of interest from the total RNA extract. The total RNA extract is preferably a total RNA extract from the same tissue and / or species from which the unknown sample is obtained. The total RNA extract may be fresh or frozen, and if frozen, may be stored as an extract or tissue sample extracted immediately before use. The term “standard curve” is used for all RNs for which graphical data can be obtained.
Also used to refer to A or cDNA dilution.

By “PCR threshold cycle” or “Ct” is intended a PCR cycle in which each PCR amplification reaction has reached a significant threshold, eg, 10 times the standard deviation of the fluorescence baseline. A "series of dilutions" is a set of dilutions of total RNA recovered from a biological sample or cDNA prepared from total RNA used as a standard from which a standard curve is generated for each gene of interest. Reverse transcription of a series of dilutions and / or PC
The results of the R amplification determine a standard curve for each gene. The members of the series may be prepared by diluting RNA or cDNA in a serial or parallel format. The RNA or cDNA standards can be from controls, sham, processed, or other categories of biological samples that contain and / or express the gene of interest.

The term “total RNA of interest” is intended to mean total RNA extracted from a sample used as a measure of the baseline conditions of the sample. The term "treated total RNA" is intended to mean total RNA extracted from a treated or treated sample whose effect on the expression of the gene of interest is being tested. The phrase "RNA equivalent" refers to the measured level of target RNA in a sample relative to a total RNA standard curve determined for the gene of interest using a reverse transcriptase-5'-exonuclease assay as described below. Used to point. The phrase "normalized RNA equivalent" refers to the RNA equivalent normalized for the amount of RNA in each reaction vessel in a 5'-exonuclease assay.

[0013] The term "treatment" is used to contemplate, but is not limited to, any manipulation of a biological sample or a patient from which the biological sample is derived, such as a physiological treatment, for example,
Pharmacological treatments, diseases, such as cardiac pressure overload hypertrophy due to aortic stenosis, exposure of an organism to a tsunami sample or patient to a chemical reagent that has or is suspected of having a physiological, biochemical, therapeutic or toxic effect. Onset of pathological conditions such as the onset or progression of cancer, heart failure, etc., surgical treatment including sham surgery, gene therapy or genetic treatment such as genetic manipulation to create knockout cells or animals or transgenic cells or animals, as described above. , And an adaptive response to any of the above-described processes.

As used herein, the terms “polynucleotide” and “oligonucleotide” refer to polydeoxyribonucleotides (including 2-deoxy-D-ribose),
Polyribonucleotides (including D-ribose), N- of purine or pyrimidine bases
Or any other type of polynucleotide that is a C-glycoside, and other polymers containing a non-nucleotide backbone, such as polyamides (eg, peptide nucleic acids (PNA
s)) and polymorpholinos (Neugen from Anti-Virals, Inc., Corvallis, Oregon)
e (trade name) polymer), and other synthetic sequence-specific nucleic acid polymers that contain nucleobases in a configuration that allows base pairing and base stacking as found in DNA and RNA Is common. There is no intended length distinction between the terms "polynucleotide" and "oligonucleotide", and these terms are used interchangeably. These terms refer only to the primary structure of the molecule. That is, these terms include, for example, 3′-deoxy-2 ′, 5′-DNA,
Oligodeoxyribonucleotide N3 '→ P5' phosphoramidate, 2'-O-alkyl substituted RNA, double- and single-stranded DNA, and double- and single-stranded RNA,
DNA: RNA hybrids and hybrids between PNAs and DNA or RNA and also include known types of modifications, eg, labels, methylations, "caps", analogs of naturally occurring nucleotides known in the art. One or more substitutions, internucleotide modifications, eg, uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), negatively charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), positively charged Those having a bond (for example, aminoalkylphosphoramidate, aminoalkylphosphotriester), those having a pendant moiety, for example, proteins (nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators What you have (for example,
Acridine, psoralen, etc.), those containing chelating agents (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (
For example, alpha-anomeric nucleic acids), as well as unmodified forms of polynucleotides or oligonucleotides.

The term “primer” is comprised of DNA and / or RNA, and / or synthetic nucleotide analogs, and is a starting point for synthesis along a complementary polynucleotide molecule under conditions suitable for the synthesis of primer extension products. Used to refer to an oligonucleotide that can act as a. Such conditions include the presence of four different deoxyribonucleotide triphosphates and at least one polymerization catalyzing agent such as reverse transcriptase or DNA polymerase. The deoxyribonucleotide is present at a suitable temperature in a suitable buffer containing the necessary cofactor. Preferably, the primer is a single-stranded oligonucleotide. As used herein, the term “target region” or “target nucleotide sequence” refers to a region contained within a gene of interest, mRN encoded by the gene of interest.
A, or the gene of interest or the complement of the mRNA encoded by the gene of interest, which is amplified and / or detected. The term "target sequence" refers to a sequence for which the probe forms a stable hybrid under the desired conditions.

As used herein, the term “hybridization probe” comprises a polynucleotide as defined above, which is complementary to the nucleic acid sequence present in the gene of interest, A reverse transcript of mRNA to be obtained, or a gene of interest or a structure containing the complement of mRNA encoded by the gene of interest. Hybridization probes may consist of DNA, and / or RNA, and / or synthetic nucleotide analogs. In addition, the probe optionally comprises 3'-phosphate, and the probe is not provided as an extension primer for DNA polymerase or reverse transcriptase. A “probe” also includes a detectable label, which does not generate a signal when the probe is intact and not degraded, but does generate a detectable signal when it leaves its complement by 5′-exonuclease digestion. A detectable label typically consists of two different fluorescent dyes. One dye is a reporter dye and the other is a quenching dye. When the probe is intact, fluorescence energy transfer occurs and the fluorescence emission of the reporter dye is absorbed by the quenching dye. During the elongation phase of the PCR cycle, the fluorescent hybridization probe is cleaved by the 5'-3 'nucleotide scission activity of the DNA polymerase. When the probe is cleaved, the reporter dye emission no longer effectively transfers to the quenching dye, resulting in an increase in the reporter dye's fluorescence emission. It will be appreciated that the hybridizing sequences need not be perfectly complementary to provide a stable hybrid. In many situations, ignoring loops of four or more nucleotides, stable hybrids are formed when less than about 10% of the bases are mismatched. Thus, the term "complementary" as used herein refers to oligonucleotides that form a stable duplex with their "complement" under assay conditions, with about 90% or more homology.

“Biological sample” as used herein refers to a sample of tissue or fluid isolated from a patient, including but not limited to plasma, serum, spinal fluid, semen, lymph, Including external fragments of skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, biopsies, and in vitro cell culture components, including but not limited to cells in cell culture media (Including conditioned media from growth, putative virus-infected cells, recombinant cells, and cellular components). A preferred use of the method of the present invention is for the detection and / or quantification of gene expression as follows: gene therapy, genetic manipulation such as generation of knockout cells or animals and generation of transgenic cells or animals; myocardial infarction or Surgical animal models such as models of pressure overload hypertrophy; clinical samples such as biopsies obtained during clinical trials or for research purposes, such as cancerous breast tissue; and the response of cultured cells and / or tissues to chemical treatment.

“Any” or “optionally” means that the condition described subsequently may or may not occur, and that the description will include instances in which the condition occurs and instances in which it does not. And For example, the phrase "possibly including a ceramic powder" means that the ceramic powder may or may not be present, and the description describes examples where ceramic powder is present and examples where ceramic powder is not present. Including. The present invention uses a quantitative reverse transcriptase polymerase chain reaction with real-time detection and a 5'-exonuclease assay to determine the expression level of the target gene. The 5'-exonuclease assay for RT-PCR is separately 5'-exonuclease.
-Known as nuclease assays, nick translation assays, etc.
olland et al., (1991) Proc. Natl. Acad. sci. USA 88: 7276-7280, Gibson et al., (19
96) Genome Res. 6: 995-1001 and Heid et al., (1996) Gemone Res. 6: 986-994. Briefly, 5 'to 3' exonuclease assays for detecting PCR products use non-extendable oligonucleotide hybridization probes. Probes are doubly labeled to monitor the results of the assay. Probes are reporter fluorescent dyes, such as fluorescent phosphoramidites, at the 5 'end, for example, 6-carboxy-fluorescein ("FAM"), 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein ("JOE"). ), Tetrachloro-6-carboxy-fluorescein (TET) and tetrachloro-6-carboxy-rhodamine (“TAMRA”). The labeled probe is P
It is designed to anneal to a region of one strand of the PCR product downstream of one of the CR primers. The 3 'end of the probe has been extended by 3'-phosphate. The probe is included with other PCR reagents, such as dNTPs,
When annealed, the DNA polymerase is degraded by the inherent 5 ′ → 3 ′ exonuclease activity. When the probe is intact, the emission of the reporter dye is quenched by the physical proximity of the reporter and the quencher fluorescent dye. However, during the elongation phase of the PCR cycle, the nucleotide cleavage activity of the DNA polymerase cleaves the hybridization probe, releasing the reporter dye from the probe.

The resulting relative increase in reporter fluorescent dye emission is monitored in real time during PCR amplification. By comparing the amount of reporter dye luminescence during PCR amplification (R) with a passive control dye, eg, 5-carboxy-X-rhodamine (ROX) (P), ΔRn
The value (R / P) is obtained. The ΔRn value reflects the amount of degraded hybridization probe. The log function is matched to the average ΔRn value, typically for the last three data points of each PCR extension cycle, and an amplification plot is generated. The relative fluorescence threshold was set based on the baseline of ΔRn during the first 10-15 cycles (
Heid et al., (1996) Genome Res. 6: 986-994). The cycle at which the PCR amplification reached a significant threshold, for example 10 times the standard deviation of the baseline, was calculated as Ct. Ct
Is proportional to the number of target copies present in the sample. Heid et al., Supra. That is, the Ct value is a quantitative measure of the copy of the target in any sample. A rapid measurement of relative changes in gene expression levels is made by using the disclosed and claimed invention to determine the value of the target gene RNA equivalent using a total RNA standard curve. Thus, there is no need for a set of reactions in which a known number of copies of the co-amplified polynucleotide are added to determine the internal control, eg, the copy number of the unknown target gene RNA. The expression level of the target gene is determined by calculating the amount of target gene RNA relative to the total RNA used for the standard curve, ie, the relative RNA equivalent.

A typical RT-PCR assay is performed as follows. Total RNA is extracted from the biological sample. If the assay includes a control biological sample and a biological sample that has been subjected to experimental treatment, stimulation or other treatment, control total RNA is extracted from the control sample and treated total RNA is extracted from the treated sample. I do. The cells or homogenized tissue are lysed by a method that disrupts the cells and preferably inactivates the ribonuclease. The RNAses are inactivated with 4M guanidinium thiocyanate and a reducing agent such as β-mercaptoethanol. A mixture of such reagents can be used to isolate intact RNA from tissues containing RNAses activity. See Sambrook et al., Pages 7.6-7.11.

[0021] Total RNA may be extracted using any method known in the art. For example, total RNA can be obtained from Glisin et al., (1974) Biochemistry 13: 2633, Ullrich et al., (1977) Scei
196: 1313, and Chomczynski et al., (1987) Anal. Biochem. 162: 156, using the guanidinium thiocyanate / cesium chloride method or Stro.
hman et al., (1977) Cell 10: 265 and McDonald et al., (1987) Meth. Enzymol. 152: 21
It may be isolated by the guanidine HCl / organic solvent method described in 9. Alternatively, RNA can be obtained from a number of commercially available kits, such as RNA STAT-60 (
Tel-Test, Inc., friendswood, TX), RNeasy Total RNA Extraction Kit (Qiagen), Tri
pure (Boehringer Mannheim Biochemicals, Indianapolis, IN), Trizol (GIBCO
Laboratories, Gaithersburg, MD), and TRI Teagent® (Molecular R
esearch Center, Inc., Cincinnati, OH).

A dilution of the total RNA extract is used to generate a standard curve. A new standard curve is generated each time the control gene is assayed using the forward and reverse primers and a hybridization probe specific for the control gene. Typically, between about 0.1 ng total RNA per reaction to about 5000 ng RNA per reaction, preferably from about 5 ng RNA per reaction tube to about 1000 ng per reaction tube.
The reaction is carried out using between the RNAs. The range of total RNA extract per reaction is:
It can be adjusted according to the relative abundance of the target gene and the amount of test RNA per reaction. That is, for relatively abundant genes, the approximate amount of total RNA extract added per reaction tube is in the low range, while for more rare transcripts, the total R
The amount of NA extract is in the high range. In addition to performing a total RNA standard curve for each gene of interest, a standard curve for housekeeping genes is optionally performed to normalize the amount of total RNA added to each reaction. A “housekeeping gene” is a gene whose expression is substantially the same as one that relatively reflects a change in response from sample to sample or from tissue to tissue or to an external stimulus. The housekeeping gene can be any RNA molecule other than the one encoded by the gene of interest, and the sample R can be used to normalize the amount of total RNA added to each reaction.
Allows for normalization of NA or any other marker. For example, GAPDH gene, G6PD gene, β-actin gene, ribosomal RNA, 36B4 RNA and the like can be used as housekeeping genes. That is, a dilution of the total RNA extract used to generate a standard curve for expression of the target gene may be, for example, GAP
DH gene expression can also be assayed. PCR threshold cycles are determined for each of the total RNA standard concentrations and, if tested, for the housekeeping gene. The Ct values so determined are plotted against log ₁₀ of the total RNA concentration in the corresponding reaction to obtain a standard curve. The equation of this curve is used to determine the value of the input RNA equivalent or the RNA equivalent for normalization.

The total RNA in each reaction tube is reverse transcribed using any naturally occurring or recombinant enzyme having reverse transcriptase activity, such as AMV reverse transcriptase, MMLV reverse transcriptase, Tth DNA polymerase and the like. The reverse transcript is then reconstituted with P, a well known in the art, in the presence of forward and reverse primers and a target-specific hybridization probe.
Amplified using CR thermocycling. The housekeeping gene reverse transcript is amplified using a PCR thermocycling method, using forward and reverse primers and a hybridization probe specific for the housekeeping gene. The 5'-exonuclease PCR assay determines DNA polymerase activity and 5 '
Use any naturally occurring or recombinant enzyme that has' -exonuclease activity and is thermally stable. The thermostable DNA polymerase, at elevated temperatures,
It retains its ability to catalyze the addition of deoxyribo-nucleotides to oligonucleotide primers based on the NA sequence. Such thermostable DNA polymerases include Taq DNA polymerase (Thermus aquaticus); Tth DNA polymerase (Thermus thermophilus HB8); and Tf1 DNA polymerase (Thermus thermophilus HB8).
mus flavus).

More specifically, PCR uses short oligonucleotide primers (generally 10-20 nucleotides in length) that match opposite ends of the desired sequence in the DNA molecule. The initial template can be RNA or DNA. When using RNA, it is first reverse transcribed into cDNA. The cDNA is then denatured using well known techniques such as heat, and the appropriate oligonucleotide primers are added in molar excess. The primer hybridizes to a complementary target nucleotide sequence, and primer extension is catalyzed using DNA polymerase in the presence of deoxynucleotide triphosphates or nucleotide analogs. The resulting product contains the corresponding primer covalently linked at the 5 'end to the newly synthesized complement of the first strand. The replicated molecule is denatured again, hybridizes to the primer, and continues until the product is fully amplified. Such a PCR method is described, for example, in US Pat. No. 4,965,18.
No. 8,800,159; 4,683,202; 4,683,195, the entire contents of which are incorporated herein by reference.

The increase in reporter dye fluorescence is monitored in real time during PCR amplification,
Used for calculating ΔRn. “ΔRn” is the increase in fluorescent signal due to reverse transcript amplification of the target or housekeeping gene, calculated by subtracting background fluorescence: ΔRn = (Rn ⁺ ) − (Rn ⁻ ), where Rn ⁺ is the ratio of the emission intensity of the reporter dye to the emission intensity of the passive reference dye in the amplification in the presence of a target reverse transcript, Rn ^- passively control dye during amplification without target reverse transcript Is the ratio of the emission intensity of the reporter dye to the emission intensity of the reporter dye. ΔRn
Is plotted against the number of amplification cycles for each of the total RNA dilution standards and each of the housekeeping gene dilution standards. Ct is determined for each of the total RNA dilution standards and each of the housekeeping gene dilution standards. The standard curve is Ct versus log ₁₀ of total RNA concentration for a series of dilutions.
Is generated for each control gene, and optionally for the housekeeping gene, and typically results in a linear standard curve. The standard curve was fitted with a linear equation, Y = MX + B for each control gene and y = mx + b for the housekeeping gene, where Y and y are Ct
Where M and m are the slopes of the straight line, and B and b are the values of Ct where the fitted straight line crosses the vertical axis.

The normalized RNA equivalent for unknown controls and / or treated samples is
It is calculated as follows from a plot of vs. [RNA]. RNA extracts of unknown controls and / or treated samples are assayed in RT-PCR assays using forward and reverse primers and hybridization probes specific for each gene of interest. Preferably, the sample RNA extract is assayed in duplicate for each gene of interest. The sample RNA equivalent is solved by solving the linear equation for X, ie, X = (Y−B) / M for the standard curve determined for the gene of interest, then for x, ie, the housekeeping gene (“ hg ”)
By solving x = (y−b) / m for A equivalents, X is determined as the total RNA in the unknown sample.
Calculated by normalizing to quantity. Thus, for example, in an assay where 0.5 μg / reaction of total RNA is added to an unknown control sample, the RNA equivalent for the unknown sample can be calculated as follows: unknown 1 (eg, control sample) In assays where 0.5 μg / reaction of total RNA is added to the unknown test treated sample, the amount of unknown RNA equivalent can be calculated as follows: Unknown 2 (eg, treated sample) To compare mRNA expressed from the gene of interest in response to treatment,
NA equivalent ₂ is compared to RNA equivalent ₁ .

A hybridization probe capable of specifically hybridizing with the forward and reverse primers and the mRNA molecule encoded by the gene of interest or its complement, and forming a stable hybrid duplex with the segment thereof; Primers can prime the extension reaction, reverse transcriptase, and / or thermostable DNA polymerase can be provided in a diagnostic kit. The kit may include other reagents and materials properly packaged or necessary or desired for a particular assay protocol, such as reagents for extracting RNA from a sample, dNTPs, and instructions for performing the assay. The reagents used in the kits described above can be provided in one or more containers, such as vials or bottles, each container containing the reverse transcriptase, dNT used in the assay.
It contains a mixture of P or dNTPs or another reagent such as a thermostable DNA polymerase. Other components known to those skilled in the art, such as buffers, controls, etc., may be included in those test kits. The kit will also include instructions for its use.

Analytical RT-PCR assays can be used to measure quantitative changes in gene expression. This method is sensitive, requires only a very small amount of tissue sample, and is applicable for rapid testing of new candidate genes. Unlike previously reported methods, this method does not require cloned cDNA, or fragments thereof, or artificial transcripts prepared from the cDNA for quantitative calibration of the assay. In addition, previously reported methods use relatively pure or produced RNA strands. In contrast, the assay uses total RNA as a standard; using such more complex standards more closely reflects the composition of the unknown sample. In addition, this assay requires only knowledge of the complementary DNA sequence of the mRNA encoded by the gene of interest to enable PCR primer and probe design. In addition, the assays disclosed and claimed herein can be used to screen a large number of candidate genes for changes or differences in expression.

Gene expression data for a very large number of genes is currently being collected using microarray-based techniques and expressed sequence tagging. Application of these techniques to expression analysis in in vitro and in vivo models of development and pathophysiology will identify a subset of genes that are uniquely associated with each biological system. Analytical P
The application of CR will provide an available means to quickly focus on the selection of markers representing molecular phenotypic fingerprints. The possibility of quantitatively measuring changes in gene expression with small amounts of total RNA means that this analytical PCR assay has widespread application to the testing of human biopsy samples. The amount of material typically obtained at biopsy does not support microarray-based expression assays; however, such samples are sufficient for use in the RT-PCR disclosed and claimed herein. The possibility of making these measurements in human samples would provide a means to identify diagnostic molecular markers associated with disease progression and exacerbation and to determine the efficacy of treatment strategies. This data also has important links to animal models of human disease.

For example, when loaded by various nerve solutions, mechanical stimuli, and cell damage,
Adult hearts respond by cardiomyocyte hypertrophy. At the cellular level, the hypertrophic response is characterized by an increase in the size of individual muscle cells without a change in cell number. Although initially compensatory, pathological transitions can occur with myocardial dysfunction and overt heart failure. Determining gene expression patterns observed in different models of hypertrophy provides clues to distinguish between adaptation and dysfunction. Such a determination enables the identification of pathways that activate different forms of cardiac hypertrophy and ultimately indicate the pathological phenotype of ventricular dilation and injury.

The most retained feature of cardiac hypertrophy is the reactivation of genes whose expression is normally restricted to the embryonic window of cardiac development. For example, atrial natriuretic peptide (
The (ANP) gene is expressed in both embryonic atrium and ventricle, but is down-regulated in the ventricle during postnatal development. However, ventricular ANP gene expression is
Immediately elicited more than 10-fold in response to hemodynamic stress, a response common to all vertebrate species tested so far. These findings, along with numerous data on other genes, have led to the hypothesis that there is a common pathway involved in the regulation of developmental gene expression and the setting of adult cardiac hypertrophy to increased load. These common pathways will in part result in a common molecular phenotype between the two tissue states. Although widely accepted, this hypothesis has not been rigorously tested,
Only a few candidate genes have been tested so far at key points in ventricular embryonic development, normal heart postnatal growth, and cardiac hypertrophy in adult hearts.

The method of measuring in vivo cardiac function in mice, combining mouse genetic and molecular genetic techniques, offers the opportunity to directly determine the effect of genotype on cardiac physiology. Based on gene knockout and transgenic methods,
Several mouse models of cardiac hypertrophy have been described, including cardiomyocyte-specific transgene expression of α1B-adrenergic receptors, activated H-ras p21, and gp1
Includes hypertrophy due to activation of 30 signaling. Dilated cardiomyopathy is also MLP and M
nSOD knockout mice. These various mouse models
Although having similar functional and pathological abnormalities to human heart failure, it is not known to what extent these genetic modifications replicate the molecular events involved in the initiation and progression of myocardial disease. A missing aspect of these experiments is the monitoring of changes in mouse genotype-based gene expression profiles, molecular phenotypes, and how these data can be compared to human cardiac physiology, pathology, and molecular phenotypes. Related to what to compare.

Using the methods disclosed herein, the gene expression profile for mouse myocardium in normal development and adaptive hypertrophy was determined. This RT-PCR assay was performed on a panel of 29 genes of tissue samples derived from three stages of cardiac growth; late embryonic ventricular development, normal postnatal heart growth, and postnatal hypertrophy following pressure overload from aortic constriction. Applied. Using this method, evidence is provided that a diverse molecular profile exists for each of the three different stages of ventricular growth. In addition, new classes of molecules that are selectively regulated in each of the three settings have been identified and their particularly potential importance in the differentiation characteristics of the three specific stages of normal and pathological heart growth and hypertrophy Suggests gender.

B. EXPERIMENTAL The practice of the present invention will employ, unless otherwise indicated, conventional techniques of synthetic organic chemistry, biochemistry, molecular biology, etc., which are within the skill of those in the art. Such techniques are explained fully in the literature. See, for example, Sambrook, Fritsh and Maniatis, Molecu
lar Cloning: A Laboratory Manual, Second Edition (1989); Origonucleotide
Synthesis (MJ Gait, Ed., 1984); Nucleic Acid Hybridization (BD Hames
And SJ Higgins, eds., 1984); and continuum, Methods in Enzymology (Academic
Press, Inc.). Although the invention has been described with reference to preferred specific embodiments thereof, the above description, as well as the following examples, are intended to be illustrative, but not limiting, of the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Temperatures are always given in degrees Celsius, and unless otherwise specified, pressures are near atmospheric.

Synthesis of ROX standards. Fluorescent standard 5'-TTT-TTT- (LAN-
(ROX) -p3 'was prepared as follows: "ROX", 5-ROX, SE (5-carboxy-X-rhodamine, succinimidyl ester) was obtained from Molecular Probes, Inc. (E
ugene, OR). “LAN”, linker arm nucleotide (amino modified C6dT), and 3′-phosphate-CPG (solid support), 46 μm / g
n Purchased from Research (Sterling, VA). The synthesis was performed on an ABI 394 DNA synthesizer on a 10 μm scale according to the following steps: (1) LAN was coupled to 3′-phosphate-CPG; (2) T6 phosphoramidite was LAN-3 ′.
(3) Cleavage of the oligonucleotide from CPG was performed in 20 ml of concentrated NH ₄ OH at room temperature for 1-2 hours; (4
) Was vortexed and the resulting mixture was centrifuged to cause the remains by removing the supernatant evaporated; and (5) remains the 0.25M of 5 ml NaHCO ₃ (pH 9 with 1N NaOH. (Adjusted to 0). 5-ROX, SE dye (25m
g / 300 μl DMSO) was added as follows: 50 μl ROX was added, mixed by stirring and left at room temperature in the dark for 45 minutes. The addition of 50 μl was repeated and left in the dark for 45 minutes. A final 50 μl addition was made and the reaction was incubated overnight at room temperature. The reaction was then divided into 10 aliquots and the Pharmacia (
Unreacted dye residues were removed through PD-10 (Piscataway, NJ). The desired fractions were combined and evaporated, and the crude product was purified by HPLC followed by polyacrylamide gel electrophoresis purification.

Synthesis of hybridization probes. Probes are available from PE, Applied Biosystems (Fo
ster City, CA) or synthesized as follows: "TAMRA", 5-T
AMRA, SE (5-carboxytetramethylrhodamine-X-rhodamine, sushi
Nimidyl ester) was purchased from Molecular Probes, Inc. (Eugene, OR).
"LAN", linker arm nucleotide (amino modified C6dT), 3'-phospho
Fate-CPG (support), 46 μm / g, and “FAM”, 5- (5′-fluorescein)
(Inphosphoramidite) was purchased from Glen Research (Sterling, VA). Synthesis is
Performed on an ABI 394 DNA synthesizer on a 0.2 μm scale as follows: (1)
Coupling LAN to 3'-phosphate-CPG in bulk (about 34 m / reaction)
(2) Oligonucleotides were LAN-based using a 0.2 μm scale.
Synthesized using standard phosphoramidite chemistry on 3'-phosphate-CPG
(3) FAM was coupled to the completed oligonucleotide (7-10).
Min coupling time). Cleavage of the oligonucleotide from CPG was performed with 1 ml of concentrated
NH₄Performed in OH at 40 ° C. for 18 hours in the dark. Vortex the mixture
The heart was separated, the supernatant was removed and evaporated to produce a residue. To precipitate the product
The residue was dissolved in 1 ml of 1 M NaCl, and 12 ml of absolute ethanol was added.
Was. The mixture was shaken vigorously and left at -20 ° C overnight. Next, the suspension is centrifuged for 6-7 minutes.
Separated and discarded supernatant. Dry orange pellet slowly under vacuum in the dark
Excess ethanol was removed and the resulting residue was treated with 0.5 ml of 0.25 M NaHCO3. ₃ (Adjusted to pH 9.0 with 1N NaOH). 5-TAMRA, S
E dye (5 mg / 60 μl in DMSO) was added as follows: 5 μl TA
MRA was added to the reaction and vortexed at room temperature in the dark for 45 minutes. 5
The addition of μl was repeated and incubated again for 45 minutes. Add the last 5 μl
The reaction was incubated overnight at room temperature. Transfer the reaction solution to Pharmacia PD-1
0 to remove unreacted dye residue, collect and evaporate the desired fractions,
The crude product was purified by HPLC followed by polyacrylamide gel electrophoresis purification.
Was.

Preparation of tissue and RNA. Adult D57B1 / 6 mice were obtained from the National Institute of Aging (Bethesda, MD) for sex and aging experiments. Mice were euthanized by cervical dislocation next to CO _2. Tissues were removed, snap frozen in liquid nitrogen five times and stored at -80 C until RNA preparation. Neonatal ventricles were obtained from Simonsen Laboratories, Inc. (Gilroy, CA). Rockman et al., (1993) Circulation 87 Supplement VII: VII-14-VI
As already described in I-21, pressure overload hypertrophy (PO
L).

RNA was extracted using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's instructions at a concentration of 0.5 μg / μl in 10-15 μg aliquots at −80 ° C. Stored. A set of probes and primers for each gene was obtained using Oligo 5.0 (National Biosciences Inc., Plymouth, MN) or Primer Expr.
Designed with either ess® (PE Applied Biosustems, Foster City, CA). The rules for designing a probe / primer set are summarized as follows:
(1) Maximum amplicon size is 500 bp, preferred range is 50-150 bp
(2) the probe has no G at the 5 ′ end, has a (G + C) content of 30-80%, and hybrids with a forward or reverse primer based on a chain having ≦ 3 consecutive Gs, Cs more than Gs. Has no formation / duplication and has a Tm = 68-70 ° C .;
(3) The primer is designed to have Tm = 58-60 ° C., and the 5 nucleotides at the 3 ′ end of the primer have only 1 to 2 G + C. The forward and reverse primers and probes used in the following examples are shown in Tables 1A, 1B and 1C, respectively.
Shown in

[0039]

[0040]

[0041]

Analytical PCR. AMV reverse transcriptase and Tf1 DNA polymerase (Promega, M
adison, WI) was used for RT-PCR.
50 μl RT-PCR reaction was performed with 10 μl AMV / Tf15x reaction buffer, 3 mM MgSO ₄ , 200 μM dNTPs, 600 nM ROX standard,
It has 5 units of AMV reverse transcriptase, 5 units of Tf1 DNA polymerase, 200 nM of probe, 100-300 nM of primer and 100 ng of total RNA. RT-PCR conditions were 48 ° C. for 45 minutes, 95 ° C. for 2 minutes, and 95 ° C. for 1 minute.
40 cycles of 5 seconds, 1 minute at 60 ° C, and 15 seconds at 72 ° C. Standard curves were obtained at each time point and genes were assayed at 400 ng, 100 ng, 25 ng, 6.25 ng per reaction of total RNA prepared from pooled adult C57BL / 6 ventricles. The amount of test RNA was reduced to 1 ng total RNA for rich genes and increased to 1 μg for rare genes, and the standard curve was adapted accordingly. The reaction was performed using a model 7700 sequence detector (PE Applied Biosystems, Foster
City, CA) and run the results in a 96-well plate.
(Applied Biosystems, Foster City, CA, USA).

Calculation of relative RNA equivalents. The amount of control RNA equivalent in each sample was calculated from a plot of Ct against log ₁₀ [RNA] (μg / reaction) based on the control RNA dilution performed for each gene. Since there is no absolute copy number, the amounts are relative RNA equivalents, relative to the pooled ventricular RNA used for the standard curve. GAP
DH was used as an internal control for normalized differences in the input sample RNA. Each sample performed the GAPDH assay at the beginning and then at the end of a series of assays. The diluted RNA can be stored at 4 ° C. for up to 3 weeks to complete the assay, during which time there is no change in GAPDH Ct values. Input RNA for each reaction
Was corrected to 100 ng based on the GAPDH standard curve. Statistical method. Statistical parameters are available from Statview® software (Abacus Con
cepts, Inc., Berkeley, CA). Two-tailed t-tests were used for comparisons between the two groups, assuming equal variances. For a number of groups, analysis of variance in post hoc analysis in the Scheffe test was utilized. To minimize the effects of unequal variance between groups, a log equivalent value of RNA of ₁₀ was employed. A p-value less than 0.05 was considered significant.

Example 1 Expression Analysis in Postnatal Myocardium The purpose of this example is to use the quantitative reverse transcriptase-polymerase chain reaction assay disclosed herein for the analysis of cardiac gene expression. The selection of genes studied was also based on differences in cardiac gene expression known from models of cardiac hypertrophy in mice or other species, to test the number of candidate genes that could affect cardiac function (Table 2). . Testing a representative category of heart genes,
Includes natriuretic peptides, embryonic contractile proteins, Ca ⁺⁺ -regulatory proteins, mitochondrial enzymes, nuclear factors, matrix proteins, and signaling molecules.

The effects of aging and gender on cardiac gene expression were tested in ventricular tissue from C57BL / 6 mice. Body weight (BW) and ventricular weight (VW) were compared between groups of young adult (3 months old) and old (18 months old) male and female (4-6 animals per group). VW or VW / BW weights did not differ between groups (data not shown), reflecting that myocardial growth is proportional to body weight. Among the 29 genes tested, α
-Only skeletal actin showed differences between the adult groups. Older males have higher levels of α-skeletal actin mRNA compared to females of the same age (3.5-fold, p = 0.003)
); This difference did not reach statistical significance in young animals (2.8-fold,
p = 0.064). Neonatal gene expression is determined from a pool of one-day-old ventricles and reflects the embryonic pattern of gene expression compared to adult data (pooled from all individual measurements representing both genders and ages) Differences specifically associated with the postnatal myocardium tissue were identified. The mRNA expression levels of the seven genes did not differ between neonates and adults, but were increased in the postnatal ventricle at 15 and suppressed at 3 compared to adults (Table 2).

[0046] Normalized RNA equivalent values are three pools of 22-28 ventricles from a one-day newborn,
And from an average of 16 animals unless otherwise noted. Values are means +-SD.

Pressure Overload Hypertrophy Gene Expression Analysis of Mouse Ventricle for Acute POL Due to Aortic Stenosis
Performed in comparison with m ventricle. In this mouse surgical model of cardiac hypertrophy, VW / BW when the left ventricle undergoes afferent hypertrophy to normalize contractile wall stress.
There is a sharp increase. Increased mechanical load from aortic stenosis is thought to be the primary stimulus present in the hypertrophic response, but wall stress from focal necrosis may also play a role. By one week after surgery, 30% increase in VW / BW ratio of 12-week-old female C57BL / 6 mice
(Table 3). MRNA from a total of 20 genes was identified as induction or suppression (Table 4).

[0048] Values are means +-SD. NPR-A (0.29), ET-1 (0.67), ETAR
(0.54), VEGF (0.64), cardiac actin (0.09), VSM actin (0.39), MLP (0.55), TGFβ1 (0.12), and GAT
No statistically significant change (p value in parentheses) was observed for A-4 (0.51).

For a direct comparison of neonatal and adult hypertrophy, statistically significant gene expression differences (
Tables 2 and 4) were graphed together in a mirror plot (FIG. 2A and FIG. 2B).
). Genes are listed on the left and data were taken from Tables 2 and 4. Statistically insignificant ratios were given a value of 1 for comparison purposes. All genes that showed a significant change in any case were plotted.

These data show that the methods disclosed and claimed herein can quantify gene expression and quantify changes in gene expression with small amounts of total RNA. The possibility of making such quantitative measurements immediately allows the assay to be used to quantify the expression of a panel of genes in an array of samples. Thus, provided herein is a method of determining the expression level of a gene. Although the preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and scope of the invention as defined in the appended claims.

[Sequence list]

[Brief description of the drawings]

Fig. 1A is a graph showing the increase in fluorescence intensity from hybridization probe degradation, a four-fold increase in total RNA extract from mouse ventricles using forward and reverse primers and a hybridization probe specific for cardiac ankyrin repeat protein. ΔRn as a function of the number of PCR cycles for each dilution series
It was expressed by. Total RNA concentration in ng / reaction tube performed in replication was 400 (solid circle),
100 (solid triangle), 25 (solid square), and 6.25 (solid diamond). Each PC
The PCR threshold cycle (Ct) at which the R amplification reaction reaches 10 times the standard deviation of the fluorescence baseline is indicated by an arrow.

FIG. 1B is a graph showing Ct determined from FIG. 1A plotted against total RNA in ng / reaction on a log scale. The linear regression equation for the points plotted in FIG. 1B is y = 24.90-3.36log (x), R = 0.997.

FIGS. 2A and 2B are graphs showing a comparison of molecular phenotypes for development and pressure overload cardiac hypertrophy (POL). Differences in gene expression for neonates versus adults (FIG 2A) and POLs versus adults (FIG 2B) were plotted as mirror image bar graphs, with induction indicated by open bars and inhibition indicated by hatched bars.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] May 4, 2000 (200.5.4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction contents]

[0021] Total RNA may be extracted using any method known in the art. For example, total RNA can be obtained from Glisin et al., (1974) Biochemistry 13: 2633, Ullrich et al., (1977) Scei
196: 1313, and Chomczynski et al., (1987) Anal. Biochem. 162: 156, using the guanidinium thiocyanate / cesium chloride method or Stro.
hman et al., (1977) Cell 10: 265 and McDonald et al., (1987) Meth. Enzymol. 152: 21
It may be isolated by the guanidine HCl / organic solvent method described in 9. Alternatively, RNA can be obtained from a number of commercially available kits, such as RNA STAT-60 (
Tel-Test, Inc., friendswood, TX), RNeasy (brand name) Total RNA extraction kit (Qiag
en), Tripure (Boehringer Mannheim Biochemicals, Indianapolis, IN), Trizo
l may be extracted using any of (trade name) (GIBCO Laboratories, Gaithersburg, MD) and TRI Teagent® (Molecular Research Center, Inc., Cincinnati, OH).

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction contents]

B. EXPERIMENTAL The practice of the present invention will employ, unless otherwise indicated, conventional techniques of synthetic organic chemistry, biochemistry, molecular biology, etc., which are within the skill of those in the art. Such techniques are explained fully in the literature. See, for example, Sambrook, Fritsh and Maniatis, Molecu
lar Cloning: A Laboratory Manual, Second Edition (1989); Origonucleotide
Synthesis (MJ Gait, Ed., 1984); Nucleic Acid Hybridization (BD Hames
And SJ Higgins, eds., 1984); and continuum, Methods in Enzymology (Academic
Press, Inc.). Although the invention has been described with reference to preferred specific embodiments thereof, the above description as well as the following examples are intended to be illustrative . Other aspects, advantages and modifications will be apparent to those skilled in the art to which this invention belongs. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Temperatures are always given in degrees Celsius, and unless otherwise specified, pressures are near atmospheric.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction contents]

RNA was extracted using RNA STAT-60 (trade name) (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's instructions and 0.5 μg / μl in 10-15 μg aliquots.
At -80 ° C. Oligo 5.0 (trade name) (National Biosciences Inc., Plymouth, MN)
) Or Primer Express® (PE Applied Biosustems, Foster City, CA). The rules for the design of a probe / primer set are summarized as follows: (1) the maximum amplicon size is 500 bp, the preferred range is 50-150 bp; (2) the probe has no G at the 5 'end. , 30-80% (
G + C) content, ≦ 3 consecutive Gs, based on strand with Cs than Gs, no hybridization / overlap with forward or reverse primer, and Tm = 68−
(3) The primer is designed to have a Tm = 58-60 ° C., and the 5 nucleotides at the 3 ′ end of the primer have only 1-2 G + C.
The forward and reverse primers and probes used in the following examples are shown in Table 1 respectively.
A, 1B and 1C.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction contents]

These data show that the methods disclosed and claimed herein can quantify gene expression and quantify changes in gene expression with small amounts of total RNA. The possibility of making such quantitative measurements immediately allows the assay to be used to quantify the expression of a panel of genes in an array of samples. Therefore, a method of determining the expression level of the gene Ru is provided herein.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (71) Applicant 460 Point San Bruno Blvd. , South San Francisco, California 94080 USA F term (reference) 4B024 AA11 AA20 CA01 CA09 CA11 CA12 HA14 HA20 4B063 QA01 QA19 QQ02 QQ03 QQ08 QQ53 QR14 QR32 QR35 QR56 QR62 QS25 QQS34 QS34

Claims (22)

[Claims] 1. A method for determining a quantitative measure of expression of a gene of interest in a biological sample by determining the normalized RNA equivalent of the gene of interest in the sample, comprising: (a) (i) A) preparing a series of reverse transcript standard dilutions from a total RNA extract with known total RNA concentration; and (ii) using a 5'-exonuclease PCR assay for each member of the series of reverse transcript standard dilutions. Amplify, the components of the assay include a forward primer, a reverse primer, a hybridization probe and a thermostable DNA polymerase, and the primers and the probe are reverse transcripts of mRNA encoded by the gene of interest or its complement Specifically hybridizes to a segment of the primer, forming a stable hybrid duplex, and the primer primes the extension reaction Bets can be, and the DNA polymerase can catalyze the primer extension reaction 5'
(Iii) monitoring the fluorescence intensity from the 5'-exonuclease PCR assay in real time during each cycle of PCR for each member of a standard dilution of a series of reverse transcripts; (Iv) threshold cycle for each member of a standard dilution of a series of reverse transcripts (
Ct), and (v) the total RNA abundance for each member of a standard dilution of a series of reverse transcripts.
Determine the RNA equivalent from a plot of Ct against g, and (vi) normalize the RNA equivalent to obtain a normalized RNA for the gene of interest.
Generating a standard curve for the gene of interest using reverse transcriptase-5'-exonuclease polymerase chain reaction (RT-PCR) by a method comprising providing an equivalent standard curve; (b) RT -A method comprising assaying the sample in PCR and determining the normalized RNA equivalent for the gene of interest in the sample from the standard curve generated in step (a). 2. The normalization of steps (a) and (vi) is performed by determining the known R of the total RNA extract.
Log of NA concentration and total RNA amount in each member of a series of reverse transcript dilutions
Based on the plot of Ct determined for the housekeeping gene for
The method of claim 1. 3. A series of reverse transcript dilutions reverse transcribe the total RNA extract to total R
2. The method of claim 1, wherein the method is prepared by providing an NA reverse transcript and diluting the total RNA reverse transcript. 4. A series of reverse transcript dilutions is prepared by diluting the total RNA extract to provide a series of total RNA extract dilutions and reverse transcribing the series of total RNA extract dilutions. The method of claim 1. 5. The method of claim 1, wherein the total RNA extract is a total RNA extract of a biological sample. 6. A process for quantitatively measuring the expression of a gene of interest in a sample by measuring the normalized RNA equivalent of the gene of interest in the first untreated and second treated sample. (A) (i) preparing a series of reverse transcript standard dilutions from a total RNA extract having a known total RNA concentration, and (ii) preparing a series of reverse transcript standard dilutions. Each member is amplified using a 5'-exonuclease PCR assay, where the components of the assay include a forward primer, a reverse primer, a hybridization probe and a thermostable DNA polymerase, and Or can specifically hybridize to a segment of the reverse transcript of the mRNA encoded by its complement to form a stable hybrid duplex, Can the primer to prime extension reaction, and the DNA polymerase can catalyze the primer extension reaction 5'
(Iii) monitoring the fluorescence intensity from the 5'-exonuclease PCR assay in real time during each cycle of PCR for each member of a standard dilution of a series of reverse transcripts; (Iv) threshold cycle for each member of a standard dilution of a series of reverse transcripts (
Ct), and (v) the total RNA abundance for each member of a standard dilution of a series of reverse transcripts.
Determine the RNA equivalent from a plot of Ct against g, and (vi) normalize the RNA equivalent to obtain a normalized RNA for the gene of interest.
Generating a standard curve for the gene of interest using reverse transcriptase-5'-exonuclease polymerase chain reaction (RT-PCR) by a method comprising providing an equivalent standard curve; (b) RT Assaying the first and second unknown samples in the PCR to determine the first and second
Determining the normalized RNA equivalent for the gene of interest in the unknown sample from the standard curve generated in step (a). 7. The normalization of steps (a) and (vi) is performed by determining the known R of the total RNA extract.
Log of NA concentration and total RNA amount in each member of a series of reverse transcript dilutions
Based on the plot of Ct determined for the housekeeping gene for
The method of claim 6. 8. A series of reverse transcript dilutions reverse transcribe total RNA extracts to total R
7. The method of claim 6, wherein the method is prepared by providing a NA reverse transcript and diluting the total RNA reverse transcript. 9. A series of reverse transcript dilutions is prepared by diluting a total RNA extract to provide a series of total RNA extract dilutions and reverse transcribing the series of total RNA extract dilutions. The method of claim 6, wherein: 10. The total RNA extract is a total RNA extract of a biological sample.
The method of claim 6. 11. The treatment is selected from the group consisting of: physiological treatment, pharmacological treatment, initiation of a pathological condition, surgical treatment, genetic treatment, combinations thereof, and an adaptive response thereto. The method of claim 6. 12. A method for quantitatively measuring the expression of a panel of genes of interest in a sample by measuring the normalized RNA equivalent of the gene of interest in the sample, comprising: (a) (i) Preparing a series of reverse transcript standard dilutions from a total RNA extract having a total RNA concentration of: (ii) amplifying each member of the series of reverse transcript standard dilutions using a 5'-exonuclease PCR assay Wherein the components of the assay include a forward primer, a reverse primer, a hybridization probe and a thermostable DNA polymerase, and the primers and probes are segments of the reverse transcript of mRNA encoded by the gene of interest or its complement. Specifically hybridizes to form a stable hybrid duplex, and the primer can prime the extension reaction. It can be, and the DNA polymerase can catalyze the primer extension reaction 5'
(Iii) monitoring the fluorescence intensity from the 5'-exonuclease PCR assay in real time during each cycle of PCR for each member of a standard dilution of a series of reverse transcripts; (Iv) threshold cycle for each member of a standard dilution of a series of reverse transcripts (
Ct), and (v) the total RNA abundance for each member of a standard dilution of a series of reverse transcripts.
Determine the RNA equivalent from a plot of Ct against g, and (vi) normalize the RNA equivalent to obtain a normalized RNA for the gene of interest.
Creating a standard curve for the gene of interest using reverse transcriptase-5'-exonuclease polymerase chain reaction (RT-PCR) by a method comprising providing an equivalent standard curve; (b) subject RT-P using primers and probes for each gene
The sample in the CR is assayed and the normalized RN for the gene of interest in the sample
A method comprising determining the A equivalent from the standard curve generated in step (a). 13. The normalization of steps (a) and (vi) is performed by determining the known RNA concentration of the total RNA extract and the low amount of total RNA in each member of a series of reverse transcript dilutions.
13. The method of claim 12, based on a plot of Ct determined for a housekeeping gene against g. 14. The series of reverse transcript dilutions is prepared by reverse transcribing a total RNA extract to provide a total RNA reverse transcript and diluting the total RNA reverse transcript. Method. 15. A series of reverse transcript dilutions are prepared by diluting a total RNA extract to provide a series of total RNA extract dilutions and reverse transcribing a series of total RNA extract dilutions. The method of claim 12, wherein: 16. The total RNA extract is a total RNA extract of a biological sample.
The method according to claim 12. 17. Quantifying the expression of a panel of genes of interest in a sample by measuring the normalized RNA equivalents of the gene of interest in the first untreated sample and the second treated sample A method for determining the effect of a treatment on a measurement comprising: (a) (i) preparing a series of reverse transcript standard dilutions from a total RNA extract having a known total RNA concentration; Each member of the dilution is amplified using a 5'-exonuclease PCR assay, wherein the components of the assay include a forward primer, a reverse primer, a hybridization probe, and a thermostable DNA polymerase, wherein the primers and probes are of interest. Stable hybrid duplex specifically hybridized to a segment of the reverse transcript of mRNA encoded by the gene or its complement The possible formation can be the primer to prime extension reaction, and the DNA polymerase can catalyze the primer extension reaction 5'
(Iii) monitoring the fluorescence intensity from the 5'-exonuclease PCR assay in real time during each cycle of PCR for each member of a standard dilution of a series of reverse transcripts; (Iv) threshold cycle for each member of a standard dilution of a series of reverse transcripts (
Ct), and (v) the total RNA abundance for each member of a standard dilution of a series of reverse transcripts.
Determine the RNA equivalent from a plot of Ct against g, and (vi) normalize the RNA equivalent to obtain a normalized RNA for the gene of interest.
Generating a standard curve for the gene of interest using reverse transcriptase-5'-exonuclease polymerase chain reaction (RT-PCR) by a method comprising providing an equivalent standard curve; and (b) The first and second unknown samples in the RT-PCR are assayed and the first and second unknown samples are assayed.
Determining the normalized RNA equivalent for the gene of interest in the unknown sample from the standard curve generated in step (a). 18. The normalization of steps (a) and (vi) is performed by determining the known RNA concentration of the total RNA extract and the low RNA amount in each member of a series of reverse transcript dilutions.
18. The method of claim 17, based on a plot of Ct determined for housekeeping genes against g. 19. The method of claim 17, wherein a series of reverse transcript dilutions are prepared by reverse transcribing the total RNA extract to provide a total RNA reverse transcript and diluting the total RNA reverse transcript. Method. 20. A series of reverse transcript dilutions is prepared by diluting a total RNA extract to provide a series of total RNA extract dilutions and reverse transcribing the series of total RNA extract dilutions. A method according to claim 17 ,. 21. The total RNA extract is a total RNA extract of a biological sample.
The method according to claim 17. 22. The treatment is selected from the group consisting of a physiological treatment, a pharmacological treatment, the onset of a pathological condition, a surgical treatment, a genetic treatment, a combination thereof, and an adaptive response thereto. The method according to claim 17.