EP1071824A2 - Analyse quantitative d'expression de gene - Google Patents

Analyse quantitative d'expression de gene

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Publication number
EP1071824A2
EP1071824A2 EP99921467A EP99921467A EP1071824A2 EP 1071824 A2 EP1071824 A2 EP 1071824A2 EP 99921467 A EP99921467 A EP 99921467A EP 99921467 A EP99921467 A EP 99921467A EP 1071824 A2 EP1071824 A2 EP 1071824A2
Authority
EP
European Patent Office
Prior art keywords
total rna
gene
dilution series
interest
reverse
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99921467A
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German (de)
English (en)
Inventor
David G. Lowe
Jill R. Schoenfeld
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Genentech Inc
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Genentech Inc
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Publication date
Application filed by Genentech Inc filed Critical Genentech Inc
Publication of EP1071824A2 publication Critical patent/EP1071824A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • PCR reverse transcriptase-polymerase chain reaction
  • the fluorescence energy transfer (“PET”) PCR assay uses a nonextendable nucleic acid probe complementary to an internal segment of the target DNA.
  • the probe is labeled with two fluorescent moieties with the property that the emission spectrum of one overlaps the excitation spectrum of the other. As a result, the emission of the first fluorophore is largely quenched by the second fluorophore.
  • the probe is present during PCR and if PCR product is made the probe becomes susceptible to degradation via the inherent 5 ' -exonuclease activity of DNA polymerase that is specific for DNA hybridized to its complement. Nucleolytic degradation of the probe allows the two fluorophores to separate in solution, which reduces quenching and increases the intensity of the emitted light. No further post-amplification processing is required when samples are measured in a fluorescent plate reader.
  • RT-PCR quantitative reverse transcriptase-polymerase chain reaction
  • RNA profiling method based on the 5 ' -exonuclease assay for reverse transcriptase polymerase chain reaction ("RT-PCR").
  • a method for determining a quantitative measure of the expression of a gene of interest in a biological sample by determining a normalized RNA equivalent for the gene of interest in the sample.
  • the method comprises, first, preparing a standard curve for the gene of interest using a reverse transcriptase-5 ' -exonuclease polymerase chain reaction (RT-PCR) .
  • RT-PCR reverse transcriptase-5 ' -exonuclease polymerase chain reaction
  • the standard curve is prepared by a method comprising (i) preparing a reverse transcript standard dilution series from a total RNA extract having a known total RNA concentration, (ii) amplifying each member of the reverse transcript standard dilution series using a 5 ' -exonuclease PCR assay, wherein the components of the assay comprise a forward primer, a reverse primer, a nonextendable FET hybridization probe and a thermostable DNA polymerase, and wherein the primers and the probe are capable of specifically hybridizing to and forming a stable hybrid duplex with a segment of the reverse transcript of mRNA encoded by the gene of interest, or its complement, the primers are capable of priming an extension reaction, and the DNA polymerase is capable of catalyzing a primer extension reaction and has 5 ' - exonuclease activity, (iii) monitoring fluorescence intensity from the 5'- exonuclease PCR assay in real time during each cycle of the PCR for
  • a method for determining the effect of a treatment on the quantitative measure of the expression of a gene of interest in a sample.
  • the method comprises preparing a standard curve for the gene of interest using the aforementioned RT-PCR assay.
  • the normalized RNA equivalent for the gene of interest in a first untreated sample and a second treated sample is determined by assaying the first and second samples in the RT-PCR and comparing the results obtained therefrom with the standard curve.
  • a method for determining a quantitative measure of the expression of a panel of genes of interest in a sample.
  • the method comprises preparing a standard curve for each of the genes of interest using the aforementioned RT-PCR assay and a separate and distinct set of a forward primer, a reverse primer and a hybridization probe for each gene of interest.
  • the normalized RNA equivalents for the genes of interest in the sample are determined by assaying the sample in the RT-PCR for each gene of interest and comparing the results obtained therefrom with the standard curve for each respective gene of interest.
  • the method comprises preparing a standard curve for each gene of interest using the aforementioned RT-PCR assay.
  • the normalized RNA equivalent for each gene of interest in the first and second samples is determined by assaying the samples in the RT-PCR and comparing the results obtained therefrom with the standard curve for each respective gene of interest.
  • kits for conducting the aforementioned method comprising (a) a forward primer, a reverse primer and a hybridization probe, which primers and probe are capable of specifically hybridizing to the reverse transcript of the target mRNA molecule or its complement and of forming a stable hybrid duplex with a segment thereof, the primers also being capable of priming an extension reaction, (b) a reverse transcriptase, (c) a thermostable DNA polymerase, (d) instructions for conducting the method and, optionally (e) other reagents necessary for conducting the reverse transcription and/or the PCR amplification reactions.
  • FIG. 1A is a graphical illustration of the increase in fluorescence intensity from hybridization probe degradation, expressed as ⁇ Rn, as a function of PCR cycle number for a four-fold serial dilution series of a total RNA extract from mouse ventricle using forward and reverse primers and a hybridization probe specific for cardiac ankyrin repeat protein.
  • concentrations of total RNA in ng/reaction tube run in duplicate were 400 (closed circles), 100 (closed triangles), 25 (closed squares), and 6.25 (closed diamonds) .
  • the PCR threshold cycle at which each PCR amplification reaction reaches ten times the standard deviation of the fluorescence baseline (Ct) is indicated by an arrow.
  • FIG. 1A is a graphical illustration of the increase in fluorescence intensity from hybridization probe degradation, expressed as ⁇ Rn, as a function of PCR cycle number for a four-fold serial dilution series of a total RNA extract from mouse ventricle using forward and reverse primers and a hybridization
  • IB is a graphical illustration of the Ct determined from FIG. 1A plotted against the amount of total RNA in ng/reaction on a log scale.
  • FIG. 2A and FIG. 2B are graphical illustrations of the comparative molecular phenotype for developmental and pressure overload cardiac hypertrophy (POL) .
  • POL developmental and pressure overload cardiac hypertrophy
  • standard curve is intended to mean a mathematical transformation of a data obtained from a graphical depiction of the results of an RT-PCR assay in which known quantities of RNA, preferably serial dilutions thereof, are assayed to compare the results obtained thereby with the results obtained for a sample containing an unknown quantity of target RNA for the purpose of quantitating the amount of target RNA in a sample.
  • a standard curve is developed for each gene of interest from a total RNA extract.
  • the total RNA extract is preferably obtained 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 as a tissue sample that may be extracted immediately before use.
  • standard curve may also be used to refer to a total RNA or cDNA dilution from which the graphical data is obtained.
  • PCR threshold cycle or “Ct” is intended the PCR cycle at which each PCR amplification reaction reaches a significant threshold, e.g., ten times the standard deviation of the fluorescence baseline.
  • a “dilution series” is a set of dilutions of total RNA recovered from a biological sample or cDNA prepared from the total RNA used as standards from which a standard curve is prepared for each gene of interest; the results of the reverse transcription and/or PCR amplification of the dilution series defines a standard curve for each gene.
  • the members of the series may be prepared by diluting the RNA or cDNA in a serial or a parallel manner.
  • the RNA or cDNA standards can be from control, sham-operated, treated, or other category of biological sample that contains and/or expresses the gene of interest .
  • control total RNA is intended to mean total RNA extracted from a sample that is used as a measure of the baseline condition of a sample.
  • treated total RNA is intended to mean total RNA extracted from a sample that has been subjected to a treatment or intervention that is being tested for its effect on the expression of the gene of interest.
  • RNA equivalent is used to refer to the measured level of target RNA in a sample relative to the total RNA standard curve determined for the gene of interest using a reverse transcriptase-5 ' -exonuclease assay, as described below.
  • normalized RNA equivalent refers to a RNA equivalent that has been normalized for the amount of RNA in each reaction vessel in the 5 ' -exonuclease assay.
  • treatment is used to intend any manipulation of a biological sample or of a subject from which a biological sample is derived, including but not limited to a physiological treatment, e.g., the induction of cardiac pressure overload hypertrophy by aortic constriction, a pharmacological treatment such as exposure to a chemical agent having or suspected of having a physiological, biochemical, therapeutic or toxic effect on the biological sample or subject, onset of a pathological state, such as the onset or progression of a disease, e.g., cancer, heart failure, or the like, surgical treatment including sham operations, genetic treatment such as gene therapy or genetic manipulations to produce a knockout cell or animal, or a transgenic cell or animal, combinations of any of the aforementioned treatments, an adaptive response to any of the above treatments, or the like.
  • a physiological treatment e.g., the induction of cardiac pressure overload hypertrophy by aortic constriction
  • a pharmacological treatment such as exposure to a chemical agent having or suspected of having a physiological, bio
  • polynucleotide and oligonucleotide shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose) , to polyribonucleotides (containing D-ribose) , to any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs) ) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oregon, as NeugeneTM polymers), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • polynucleotide and “oligonucleotide,” and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include, for example, 3 ' -deoxy- 2 ' , 5 ' -DNA, oligodeoxyribonucleotide N3 ' ⁇ P5 ' phosphoramidates , 2 ' -O-alkyl- substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
  • primer is used to refer to a oligonucleotide composed of DNA, and/or RNA, and/or synthetic nucleotide analogs, and is capable of acting as a point of initiation of synthesis along a complementary polynucleotide molecule under conditions suitable for synthesis of a primer extension product. Such conditions include the presence of four different deoxyribonucleotide triphosphates and at least one polymerization-catalyzing agent such as a reverse transcriptase or a DNA polymerase. The deoxyribonucleotides are present in a suitable buffer, including necessary cofactors, at a suitable temperature.
  • a primer is a single- stranded oligonucleotide.
  • target region refers to a region contained within the gene of interest, the mRNA encoded by the gene of interest, a reverse transcript of the mRNA encoded by the gene of interest, or the complement of the gene of interest or the mRNA encoded by the gene of interest, that is to be amplified and/or detected.
  • target sequence refers to a sequence with which a probe will form a stable hybrid under desired conditions .
  • hybridization probe refers to a structure comprised of a polynucleotide, as defined above, that contains a nucleic acid sequence complementary to a nucleic acid sequence present within the gene of interest, a reverse transcript of the mRNA encoded by the gene of interest, or the complement of the gene of interest or the mRNA encoded by the gene of interest.
  • the hybridization probe may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.
  • a probe is optionally comprised of a 3 ' -phosphate such that the probe does not serve as an extension primer for DNA polymerase or reverse transciptase.
  • a "probe” also comprises a detectable label which, when the probe is intact and undegraded does not produce a signal and when released from its complement by 5 ' -exonuclease digestion, gives rise to a detectable signal.
  • the 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, fluorescent energy transfer occurs and the reporter dye fluorescence emission is absorbed by the quenching dye.
  • the fluorescent hybridization probe is cleaved by the 5 '-3' nucleolytic activity of the DNA polymerase. On cleavage of the probe, the reporter dye emission is no longer transferred efficiently to the quenching dye, resulting in an increase of the reporter dye fluorescence emission.
  • hybridizing sequences need not have perfect complementarity to provide stable hybrids. In many situations, stable hybrids will form where fewer than about 10% of the bases are mismatches, ignoring loops of four or more nucleotides . Accordingly, as used herein the term “complementary” refers to an oligonucleotide that forms a stable duplex with its "complement” under assay conditions, generally where there is about 90% or greater homology.
  • a "biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, biopsies and also to samples of in vi tro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components) .
  • Preferred uses of the present method are in detecting and/or quantitating gene expression as follows: genetic manipulations such as gene therapy, production of a knockout cell or animal and production of a transgenic cell or animal; surgical animal models such as models of myocardial infarction or pressure overload hypertrophy; clinical samples such as biopsies obtained during clinical trials or for research purposes, e.g., cancerous breast tissue; and responses of cultured cells and/or tissues to chemical treatment.
  • "Optional” or “optionally” means that the subsequently described circumstance may or may not occur, and that the description includes instances in which said circumstance occurs and instances in which it does not.
  • the phrase “optionally including a ceramic powder” means that a ceramic powder may or may not be present and that the description includes both the instance when the ceramic powder is present and the instance when the ceramic powder is not present .
  • the present invention used quantitative reverse transcriptase polymerase chain reaction using real time detection and the 5 ' -exonuclease assay to determine the level of expression of a target gene.
  • the 5'—3' exonuclease assay for detecting PCR products uses a nonextendable oligonucleotide hybridization probe.
  • the probe is doubly labeled to monitor the results of the assay.
  • the probe is labeled with a reporter flourescent dye, i.e., a fluorescent phosphoramidite, at the 5' end, e.g., 6-carboxy-fluorescein ("FAM”), 2, 7-dimethoxy-4, 5-dichloro-6-carboxy- fluorescein (“JOE”), and tetrachloro-6-carboxy-fluorescein (“TET”), and a quencher fluorescent dye, e.g., 6-carboxy-tetramethyl rhodamine (“TAMRA").
  • FAM 6-carboxy-fluorescein
  • JOE 5-dichloro-6-carboxy- fluorescein
  • TET tetrachloro-6-carboxy-flu
  • the labeled probe is designed to anneal to a region of one strand of the PCR product downstream from one of the PCR primers.
  • the 3' end of the probe is blocked from extension by a 3 ' -phosphate .
  • the probe is included with the other PCR reagents, e.g., dNTPs, and, when annealed, is degraded by the 5 '—3' exonuclease activity inherent in the DNA polymerase.
  • the probe is intact the reporter dye emission is quenched owing to the physical proximity of the reporter and quencher fluorescent dyes.
  • the nucleolytic activity of the DNA polymerase cleaves the hybridization probe and releases the reporter dye from the probe.
  • the resulting relative increase in reporter fluorescent dye emission is monitored in real time during PCR amplification.
  • a comparison of the amount of reporter dye emission (R) with a passive reference dye, e.g., 5- carboxy-X-rhodamine (ROX) (P) during the PCR amplification generates a ⁇ Rn value (R/P) .
  • the ⁇ Rn value reflects the amount of hybridization probe that has been degraded.
  • An exponential function may be fit to the mean ⁇ Rn values of, typically, the last three data points of each PCR extension cycle, generating an amplification plot.
  • a relative fluorescent emission threshold is set based on the baseline of the ⁇ Rn during the first 10-15 cycles ( see Heid et al. (1996) Genome Res .
  • the Ct value is a quantitative measurement of the copies of the target in any sample.
  • Rapid measurement of relative changes in gene expression levels can be achieved using the invention disclosed and claimed herein by determining target gene RNA equivalent values using total RNA standard curves.
  • an internal control i.e., a set of reactions to which a known number of copies of a co-amplified polynucleotide is added, to determine the number of copies of the unknown target gene RNA.
  • the level of expression of the target gene is determined by calculating the amount of target gene RNA relative to the total RNA used for the standard curve, i.e., the relative RNA equivalent .
  • a typical RT-PCR assay is conducted as follows. Total RNA is extracted from a biological sample . If an assay includes a control biological sample and a biological sample subject to an experimental treatment, stimulus or other intervention, control total RNA is extracted from the control sample and treated total RNA is extracted from the treated sample.
  • RNAses are inactivated by 4 M guanidinium thiocyanate and reducing agents such as ⁇ -mercaptoethanol.
  • RNAse activity can be isolated from tissues that contain RNAse activity. See Sambrook et al., pages 7.6-7.11.
  • Total RNA may be extracted from using any method known in the art.
  • total RNA may be isolated using the guanidinium thiocyanate/cesium chloride method described by Glisin et al . (1974) Biochemistry 13:2633, Ullrich et al . (1977) Science 196:1313, and Chomczynski et al. (1987) Anal. Biochem. 162:156 or by the guanidine HCl/organic solvents method described in Strohman et al . (1977) Cell 10:265 and McDonald et al. (1987) Meth. Enzymol . 152:219.
  • RNA may be extracted using any of a number of commercially available kits, for example, RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) , the RNeasy total RNA extraction kit (Qiagen) , Tripure (Boehringer Mannheim Biochemicals, Indianapolis, IN) , Trizol (GIBCO Laboratories, Gaithersburg, MD) , and TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH).
  • RNA STAT-60 Tel-Test, Inc., Friendswood, TX
  • the RNeasy total RNA extraction kit Qiagen
  • Tripure Boehringer Mannheim Biochemicals, Indianapolis, IN
  • Trizol Githersburg, MD
  • TRI Reagent® Molecular Research Center, Inc., Cincinnati, OH
  • Dilutions of the total RNA extracts are used to prepare a standard curve.
  • a new standard curve is generated each time a gene of interest is assayed using forward and reverse primers and a hybridization probe specific for the gene of interest.
  • reactions are conducted using between about 0.1 ng total RNA per reaction to about 5000 ng RNA per reaction, preferably between about 5 ng RNA per reaction tube to about 1000 ng RNA per reaction tube.
  • the range of total RNA extract per reaction can be adjusted depending on the relative abundance of the target gene and the amount of test
  • RNA per reaction For relatively abundant genes about the amount of total RNA extract added per reaction tube will be at the lower range of amounts while for more rare transcripts the amount of total RNA extract will be at the higher range of amounts.
  • a standard curve is optionally run for a housekeeping gene to normalize for the amount of total RNA added to each reaction.
  • a "housekeeping gene” is a gene the expression of which is substantially the same from sample to sample or from tissue to tissue, or one that is relatively refractory to change in response to external stimuli .
  • a housekeeping gene can be any RNA molecule other than that encoded by the gene of interest that will allow normalization of sample RNA or any other marker that can be used to normalize for the amount of total RNA added to each reaction.
  • the GAPDH gene, the G6PD gene, the ⁇ -actin gene, ribosomal RNA, 36B4 RNA, or the like may be used as a housekeeping gene.
  • the dilutions of total RNA extract used to prepare the standard curve for the expression of the target gene may also be assayed for the expression of, for example, the GAPDH gene.
  • the PCR threshold cycle is determined for each of the total RNA standard concentrations and, when assayed, for the housekeeping gene.
  • the Ct values thus determined are plotted against the log 10 of the concentration of total RNA in the corresponding reaction to obtain a standard curve.
  • the equation of this curve is used to determine equivalent values of input RNA for normalization or to determine RNA equivalents.
  • RNA in each reaction tube is reverse transcribed using any naturally occurring or recombinant enzyme that has reverse transcriptase activity, e.g., AMV reverse transcriptase, MMLV reverse transcriptase, Tth DNA polymerase, and the like.
  • the reverse transcripts are then amplified in the presence of target-specific forward and reverse primers and a target-specific hybridization probe using PCR thermocycling methods well known in the art.
  • the housekeeping gene reverse transcripts are amplified using forward and reverse primers and hybridization probes specific to the housekeeping gene using PCR thermocycling method.
  • the 5 ' -exonuclease PCR assay uses any naturally occurring or recombinant enzyme that has DNA polymerase activity and 5 ' -exonuclease activity and that is thermally stable.
  • a thermostable DNA polymerase is a DNA polymerase that, at elevated temperatures, maintains the ability to catalyze the addition of deoxyribo-nucleotides to an oligonucleotide primer based on a template DNA sequence.
  • thermostable DNA polymerases examples include: Tag DNA polymerase ( Thermus agxiaticus) ; Tth DNA polymerase ( Thermus thermophilus HB8 ; and Tfl DNA polymerase ( Thermus flavus) .
  • PCR employs short oligonucleotide primers (generally 10-20 nucleotides in length) that match opposite ends of a desired sequence within a DNA molecule.
  • the initial template can be either RNA or DNA. If RNA is used, it is first reverse transcribed to cDNA. The cDNA is then denatured, using well-known techniques such as heat, and appropriate oligonucleotide primers are added in molar excess . Primers hybridize 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 includes the respective primers at their 5 '-termini, covalently linked to the newly synthesized complements of the original strands.
  • the replicated molecule is again denatured, hybridized with primers, and so on, until the product is sufficiently amplified.
  • Such PCR methods are described in e . g. , U.S. Patent Nos. 4,965,188; 4,800,159; 4,683,202; 4,683,195; incorporated herein by reference in their entireties .
  • the increase in reporter dye fluorescence emission is monitored during the PCR amplification in real time and used to calculate the ⁇ Rn.
  • the ⁇ Rn is plotted versus the amplification cycle number for each of the total RNA dilution standards and for each of the housekeeping gene dilution standards.
  • a Ct is determined for each of the total RNA dilution standards and for the housekeeping gene dilution standards.
  • a standard curve is prepared for each gene of interest and, optionally, for a housekeeping gene, by plotting the Ct against the log 10 of the concentration of total RNA for the dilution series to yield a typically linear standard curve.
  • Normalized RNA equivalents for unknown control and/or treated samples are calculated from the plots of Ct vs [RNA] as follows.
  • the unknown control and/or treated sample RNA extract is assayed in the RT-PCR assay using forward and reverse primers and a hybridization probe specific for each gene of interest.
  • the sample RNA extract is assayed in replicate for each gene of interest .
  • Unknown 1 e.g., control sample
  • RNA equivalent i JLO x 0.5 ⁇ g/rxn
  • the amount of unknown RNA equivalent may be calculated as follows : Unknown 2 (e.g. treated sample)
  • RNA equivalent 2 __ * 0.5 ⁇ g/rxn
  • RNA equivalent 2 is compared with RNA equivalent ⁇ Forward and reverse primers and a hybridization probe capable of specifically hybridizing to the mRNA molecule encoded by the gene or genes of interest, or the complement thereof, and capable of forming a stable hybrid duplex with a segment thereof, the primers also being capable of priming an extension reaction, a reverse transcriptase, and/or a thermostable DNA polymerase can be provided in diagnostic kits.
  • the kit also may contain other suitably packaged reagents and materials needed or desirable for the particular assay protocol, for example, reagents for extracting RNA from a sample, dNTPs as well as instructions for performing the assay.
  • reagents employed in the above kit can be provided in one or more containers such as vials or bottles, with each container containing a separate reagent such as a reverse transcriptase, a dNTP or a cocktail of dNTPs, or a thermostable DNA polymerase employed in the assay.
  • a separate reagent such as a reverse transcriptase, a dNTP or a cocktail of dNTPs, or a thermostable DNA polymerase employed in the assay.
  • Other components such as buffers, controls, and the like, known to those of ordinary skill in art, may be included in such test kits.
  • the kits will also include instructions for the use thereof .
  • An analytical RT-PCR assay can be used to measure quantitative changes in gene expression. This method is sensitive, requires very little tissue sample, and is adaptable to rapidly testing new candidate genes. In contrast to previously reported methods, the present method does not require cloned cDNA, or a fragment thereof, or an artificial transcript prepared from cDNA for quantitative calibration of the assay. Furthermore, previously reported methods use relatively pure or purified RNA standards. By contrast, the present assay uses total RNA as a standard; the use of such a more complex standard more closely reflects the composition of an unknown sample.
  • the assay requires only knowledge of the DNA sequence complementary to the mRNA encoded by the gene of interest to enable PCR primer and probe design.
  • the assay method disclosed and claimed herein can be used to screen numerous candidate genes for changes or differences in expression.
  • Gene expression data on a very large number of genes is currently being gathered using microarray-based technologies and expressed sequence tag approaches.
  • Application of these technologies to expression analysis in in vitro and in vivo models of development and pathophysiology will identify subsets of genes uniquely associated with each biological system.
  • Application of analytical PCR should provide an accessible means to rapidly focus on select markers that represent a fingerprint of the molecular phenotype.
  • RNA sample The ability to quantitatively measure changes in gene expression with small amounts of total RNA means that this analytical PCR assay will be widely applicable to testing human biopsy samples.
  • the amount of material typically obtained in a biopsy does not support a microarray-based expression assay; however, such sample is sufficient to be used in the RT-PCR disclosed and claimed herein.
  • the ability to make these measurements in human samples will provide a means to identify diagnostic molecular markers associated with disease progression and prognosis, and to determine the effect of treatment strategies. This data will also provide an important link to animal models of human disease .
  • the adult heart responds by cardiac muscle cell hypertrophy.
  • the hypertrophic response is characterized by an increase in the size of individual myocytes without a concomitant change in cell number.
  • a pathological transition can occur accompanied by cardiac muscle dysfunction and overt heart failure.
  • a determination of the patterns of gene expression observed in different models of hypertrophy would provide clues to distinguish between adaptation and dysfunction. Such a determination would permit the identification of the pathways which activate different forms of cardiac hypertrophy, and which ultimately drive the pathological phenotype of cardiac chamber dilation and failure .
  • Atrial natriuretic peptide (ANP) gene is expressed in both the embryonic atrial and ventricular chambers, but is down-regulated in the ventricle during normal post-natal development.
  • ventricular ANP gene expression is rapidly induced by greater than 10-fold in response to hemodynamic loading, a response common to all vertebrate species thus far examined.
  • a fluorescent standard 5 ' -TTT-TTT- (LAN-ROX) - p3 ' which is included in each PCR reaction, was prepared as follows: "ROX”, 5-ROX, SE (5-carboxy-X-rhodamine, succinimidyl ester) was purchased from Molecular Probes, Inc. (Eugene, OR). "LAN”, the linker arm nucleotide (amino- modifier C6dT) , and 3 ' -Phosphate-CPG (solid phase support), 46 ⁇ m/g, were purchased from Glen Research (Sterling, VA) .
  • Synthesis was carried out on an ABI 394 DNA Synthesizer at 10 ⁇ m scale according to the following steps: (1) LAN was coupled to the 3 ' -Phosphate-CPG; (2) T6 Phosphoramidite was coupled to the LAN-3 ' -Phosphate-CPG; (3) cleavage of the oligonucleotide from the CPG was carried out at room temperature for 1-2 hours in 20 ml of concentrated NH OH; (4) the resultant mixture was vortexed and centrifuged and the supernatant removed and evaporated to yield a residue; and (5) the residue was dissolved in 5 ml of 0.25 M NaHC03 (adjusted to pH 9.0 with IN NaOH) .
  • 5-ROX, SE dye 25 mg/300 ⁇ l DMSO was added as follows: 50 ⁇ l of ROX was added, mixed by agitation and left in the dark at room temperature for 45 minutes. The 50 ⁇ l addition was repeated and left in the dark for 45 minutes. A final 50 ⁇ l addition was made and the reaction was incubated overnight at room temperature. The reaction was then divided into 10 aliquots and passed through Pharmacia (Piscataway, NJ) PD-10 columns to remove unreacted dye residue. The desired fractions were combined, evaporated, and the crude product was purified on HPLC followed by polyacrylamide gel electrophoresis purification.
  • Probes were either purchased from PE, Applied Biosystems (Foster City, CA) or synthesized as follows: "TAMRA” is 5-TAMRA, SE (5-carboxytetramethylrhodamine-X-rhodamine, succinimidyl ester) , was purchased from Molecular Probes, Inc. (Eugene, OR) .
  • LAN linker arm nucleotide (Amino-Modifier C6dT) , 3 ' -Phosphate-CPG (Support), 46 ⁇ m/g, and "FAM”, 5- (5' -fluorescein phosphoramidite) were purchased from Glen Research (Sterling, VA) .
  • Synthesis was carried out on an ABI 394 DNA Synthesizer at 0.2 ⁇ m scale as follows: (1) LAN was coupled to the 3 ' -Phosphate-CPG in bulk (approximately 34 ⁇ m/reaction) ; (2) using a 0.2 ⁇ m scale, the oligonucleotide was synthesized on the LAN-3 ' -Phosphate-CPG using standard phosphoramidite chemistry; and (3) FAM was coupled to the completed oligonucleotide (7-10 minute coupling time) . Cleavage of the oligonucleotide from the CPG was carried out in the dark at 40°C for 18 hours in 1 ml of concentrated NH OH.
  • 5-TAMRA SE dye (5 mg in 60 ⁇ l DMSO) was added as follows: 5 ⁇ l of TAMRA was added to the reaction, vortexed and left in the dark at room temperature for 45 minutes. The 5 ⁇ l addition was repeated and again incubated for 45 minutes. A final 5 ⁇ l addition was made and the reaction was incubated overnight at room temperature . The reaction solution was passed through a Pharmacia PD-10 Column to remove unreacted dye, the desired fraction (s) were collected, combined and evaporated, and the crude product was purified on HPLC followed by polyacrylamide gel electrophoresis purification.
  • mice were obtained from The National Institute for Aging (Bethesda, MD) . Mice were euthanized with C02 to effect followed by cervical dislocation. Tissues were removed and snap frozen in liquid nitrogen within five minutes, and stored at -80°C until RNA preparation. Neonatal ventricles were obtained from Simonsen Laboratories, Inc. (Gilroy, CA) . Pressure overload hypertrophy (POL) was induced by transverse aortic constriction as previously described by Rockman et al. (1993) Circulation 87 Supplement VII:VII-14-VII-21.
  • POL Pressure overload hypertrophy
  • the forward and reverse primers and probes used in the examples below are listed in Tables 1A, IB and 1C, respectively.
  • VSM Actin AAA CAG GAA TAC GAC GAA G 14 ⁇ Actin AGA TTA CTG CTC TGG CTC CTA 15 a MHC CCA ATG AGT ACC GCG TGA A 16 ⁇ MHC ATG TGC CGG ACC TTG GAA 17
  • VEGF CAC AGG ACG GCT TGA AGA TGT 40
  • VSM Actin CAG GAA TGA TTT GGA AAG GA 44 ⁇ Actin CAA AGA AAG GGT GTA AAA CG 45
  • VSM Actin ACT TAG AAG CAT TTG CGG TGG ACG A 74 ⁇ Actm CGG ACT CAT CGT ACT CCT GCT TGC TG 74 a MHC TGA CCC GAG GCA AGC CT CCT ACA 76 ⁇ MHC CAG CGT TCT GTC AAT GAC CTC ACC AG 77
  • ANALYTICAL PCR The Access RT-PCR System, using AMV reverse transcriptase and Tfl DNA polymerase , ( Promega, Madison, WI) was used for RT- PCR .
  • 50 ⁇ l RT-PCR reactions have 10 ⁇ l of AMV/ fl 5x Reaction Buffer , 3 mM MgS0 4 , 200 ⁇ M dNTPs , 600 nM ROX standard, 5 Units AMV Reverse Transcriptase , 5 Units of Tfl DNA polymerase , 200 nM probe , 100 -300 nM primers and 100 ng of total RNA.
  • RT-PCR conditions are 45 minutes at 48°C, 2 minutes at 95°C, and 40 cycles of 15 seconds at 95°C, 1 minute at 60°C, and 15 seconds at 72 C C.
  • Standard curves were obtained each time a gene was assayed using total RNA prepared from pooled adult C57BL/6 ventricles with 400 ng, 100 ng, 25 ng, 6.25 ng per reaction.
  • the amount of test RNA may be reduced to 1 ng of total RNA for abundant genes or increased to 1 ⁇ g for rarer transcripts, with standard curves adjusted accordingly.
  • RNA EQUIVALENTS CALCULATION OF RELATIVE RNA EQUIVALENTS.
  • the equivalent amount of control RNA in each sample is calculated from a plot of Ct versus log ⁇ o [RNA] ( ⁇ g/reaction) based on the control RNA dilution run for each gene. Since there is no absolute copy number, the quantity is relative to the pooled ventricular RNA used for the standard curve, the relative RNA equivalent.
  • GAPDH was used as an internal control to normalize differences in input sample RNA. Each sample was run in a GAPDH assay at the beginning of the series of assays and then at the end. The diluted RNA can be stored at 4°C for up to three weeks to allow for assay completion, during which time there is no change in the GAPDH Ct value. The quantity of input RNA in each reaction was corrected to 100 ng based on the GAPDH standard curve.
  • the purpose of this experiment was to use the quantitative reverse transcriptase-polymerase chain reaction assay disclosed herein to analyze cardiac gene expression.
  • cardiac function The selection of genes studied was based on known differences in cardiac gene expression from models of cardiac hypertrophy in the mouse and other species, and also to test a number of candidate genes which may impact cardiac function (Table 2) . Representative categories of cardiac genes were examined, including natriuretic peptides, embryonic contractile proteins, Ca++-regulatory proteins, mitochondrial enzymes, nuclear factors, matrix proteins, and signal transduction molecules.
  • Neonatal gene expression was determined from pools of 1 day-old ventricles and was compared to adult data (pooled from all individual measurements, representing both sexes and ages) to identify differences specifically associated with the postnatal myocardium, a tissue that reflects embryonic patterns of gene expression. mRNA expression levels for 7 genes showed no difference between neonate and adult myocardium, 15 were elevated in the postnatal ventricles and 3 were repressed compared to adults (Table 2) .
  • RNA equivalent values were obtained from three pools of 22-28 ventricles from Day 1 neonates and 16 animals for the adult average except where noted. Values are the mean ⁇ SD.
  • Example 2 Pressure Overload Hypertrophy Gene expression analysis of mouse ventricles in response to acute POL by aortic constriction was performed in comparison to sham ventricles.
  • this mouse surgical model of cardiac hypertrophy there is an acute increase in VW/BW as the left ventricle undergoes concentric hypertrophy to normalize systolic wall stress.
  • Increased mechanical load from aortic constriction is thought to be the primary stimulus underlying the hypertrophic response, however wall stress due to focal necrosis may also play a role.
  • By one-week post-surgery there is a 30% increase in the VW/BW ratio of 12-week old female C57BL/6 mice (Table 3) .
  • mRNAs from a total of 20 genes were identified as induced or repressed (Table 4) .
  • NPR-A 0.29), ET-1 (0.67), ETAR (0.54), VEGF (0.64), a Cardiac Actin (0.09), VSM Actin (0.39), MLP ( 0.55), TGF ⁇ l (0.12), and GATA-4 (0.51).

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Abstract

L'invention concerne un procédé servant à déterminer le niveau d'expression d'un gène intéressant, ou d'un groupe de gènes intéressants, dans un échantillon biologique. Le procédé comporte la préparation d'une courbe normalisée pour chaque gène intéressant, mise en oeuvre par le dosage d'une série de dilutions préparées à partir d'ARN complet dans un test d'amplification par PCR de 5'-exonucléase à la transcriptase inverse. Un cycle de seuil (Ct) pour chaque élément de la série transcrite inverse - amplifiée est déterminé et tracé par opposition au relevé de la quantité d'ARN complet de la série de dilutions. Le tracé est utilisé pour déterminer un ARN équivalent à partir duquel on détermine l'ARN normalisé équivalent du gène intéressant de l'échantillon. Le dosage peut être utilisé pour déterminer l'effet d'un traitement de l'échantillon sur le niveau d'expression d'un gène intéressant ou d'un groupe de gènes intéressants.
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US6692916B2 (en) 1999-06-28 2004-02-17 Source Precision Medicine, Inc. Systems and methods for characterizing a biological condition or agent using precision gene expression profiles
US6960439B2 (en) 1999-06-28 2005-11-01 Source Precision Medicine, Inc. Identification, monitoring and treatment of disease and characterization of biological condition using gene expression profiles
US6440706B1 (en) 1999-08-02 2002-08-27 Johns Hopkins University Digital amplification
EP1268853A2 (fr) * 2000-03-20 2003-01-02 Forinnova AS Procede de determination de la duplication ou de la suppression d'un adn genomique heterozygote
DE10045521A1 (de) 2000-03-31 2001-10-04 Roche Diagnostics Gmbh Nukleinsäureamplifikationen
US6691041B2 (en) 2000-03-31 2004-02-10 Roche Molecular Systems, Inc. Method for the efficiency-corrected real-time quantification of nucleic acids
WO2002016940A2 (fr) * 2000-08-23 2002-02-28 Genome Therapeutics Corporation Identification rapide de cibles assistee par une technique genomique
GB2370351A (en) * 2000-12-22 2002-06-26 Secr Defence Brit Nucleic acid quantitation
US6964850B2 (en) 2001-11-09 2005-11-15 Source Precision Medicine, Inc. Identification, monitoring and treatment of disease and characterization of biological condition using gene expression profiles
EP1456648B8 (fr) * 2001-11-20 2009-07-08 EXACT Sciences Corporation Procedes et dispositifs de preparation automatisee d'echantillon
AU2003211947A1 (en) 2002-02-20 2003-09-09 Sysmex Corporation PRIMERS FOR NUCLEIC ACID AMPLIFICATION IN DETECTING HOUSEKEEPING GENE mRNA AND TEST METHOD USING THESE PRIMERS
FR2842211A1 (fr) * 2002-07-09 2004-01-16 Inst Necker Procede et moyens pour l'analyse quantitative du nombre des molecules d'arnm codant pour des genes differents dans des cellules
US7822556B2 (en) 2003-04-29 2010-10-26 The Jackson Laboratory Expression data analysis systems and methods
US7881873B2 (en) 2003-04-29 2011-02-01 The Jackson Laboratory Systems and methods for statistical genomic DNA based analysis and evaluation
FR2874026B1 (fr) * 2004-08-04 2006-11-17 Centre Nat Rech Scient Cnrse QUANTIFICATION DES ARNm DE PROTEINES D'INTERET AU MOYEN D'UNE REACTION RT-PCR EN TEMPS REEL
JP2007124983A (ja) * 2005-11-07 2007-05-24 National Institute Of Advanced Industrial & Technology 遺伝子発現量を規格化するための標準遺伝子
US8346485B2 (en) 2008-11-25 2013-01-01 Quest Diagnostics Investments Incorporated Methods and apparatuses for estimating initial target nucleic acid concentration in a sample by modeling background signal and cycle-dependent amplification efficiency of a polymerase chain reaction

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