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WO1998035058A2 - Procede ameliore de detection et de quantification de molecules d'acides nucleiques - Google Patents

Procede ameliore de detection et de quantification de molecules d'acides nucleiques Download PDF

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Publication number
WO1998035058A2
WO1998035058A2 PCT/US1998/001471 US9801471W WO9835058A2 WO 1998035058 A2 WO1998035058 A2 WO 1998035058A2 US 9801471 W US9801471 W US 9801471W WO 9835058 A2 WO9835058 A2 WO 9835058A2
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rna
nucleic acid
molecules
sequence
pcr
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PCT/US1998/001471
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WO1998035058A3 (fr
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James D. Thompson
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Ribozyme Pharmaceuticals, Inc.
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Priority to AU63173/98A priority Critical patent/AU6317398A/en
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Publication of WO1998035058A3 publication Critical patent/WO1998035058A3/fr

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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q1/6851—Quantitative amplification

Definitions

  • This invention relates to improved methods for detecting and quantifying desired nucleic acid molecules from a complex mixture of nucleic acids .
  • RNA abundance is the most widely used parameter for quantifying gene expression.
  • the two techniques most widely used to assay mRNA production in cells or tissues are Northern analysis (Maniatis et al , 1982, Molecular Cloning— A Labora tory Manual , Cold Spring Harbor Press) and ribonuclease protection assays (RPAs) (Berk et al , 1977, Cell 12, 721; Myers et al , 1985, Science 230, 1242) .
  • RPAs ribonuclease protection assays
  • House-keeping genes are genes that are expressed in a wide variety of cell types, whose levels of expression do not change appreciably in response to external stimuli and/or whose levels of expression are comparable between different cell types. Coanalysis of experimental or desired mRNAs with mRNAs from housekeeping genes serves to control for potential variability in sample RNA integrity and potential loading differences between different samples. Examples of house-keeping genes typically used as controls in Northern and RPA analyses are structural genes such as ⁇ -actin, major histocompatibility genes such as ⁇ 2 microglobulin and genes encoding enzymes that are involved in important metabolic pathways such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the glycolysis pathway.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • RNAs expressed at less than 100 copies per cell are difficult to detect in total cellular RNA with either of these techniques. This is because mRNAs comprise less than 5% of the RNA found in most cell types.
  • poly (A) +mRNAs must be purified from the total RNA in order to obtain sufficient amounts of the target RNA to analyze by Northern or RPA.
  • the amounts of RNA required (usually over 100 micrograms) can be prohibitive when cell or tissue types being studied are rare, precious or scarce.
  • the problem of analyzing low abundance mRNAs was partially solved with the development of the quantitative polymerase chain reaction (PCR) techniques
  • the copy number of an RNA sequence in a given sample is quantified by titrating known amounts of a synthetic competitor into the sample RNA.
  • the synthetic competitor is identical to the target sequence except for minor mutations used to distinguish its amplification product from that of the intended target mRNA sequence.
  • Amplification product from the competitor is sufficiently similar to the product from the intended target to allow the two products to hybridize to form heterodimers .
  • the amount of input competitor required to inhibit amplification of the intended target by 50% is generally used to determine the amount of target mRNA originally present in the sample.
  • Drawbacks of the QC-PCR technique are that it is not readily adaptable to co- analysis of an internal house-keeping gene to control for differences in loading and integrity of the sample RNA and it is labor intensive since up to ten PCR reactions are usually required to generate a titration curve covering a broad enough range of input competitor to accurately quantify target mRNAs.
  • the rate of accumulation of product from the intended target RNA is compared to the rate of accumulation of product from a control mRNA that is amplified from the same sample RNA but in a separate amplification reaction.
  • differences in RNA loading and integrity can be controlled.
  • manual quantification of product accumulation throughout the course of PCR amplification process can be labor intensive.
  • a device that automatically monitors product accumulation has been introduced to the market recently (Gibson et al . , 1996, Genome Res . , 6, 995; Chiang et al , 1996, Genome Res . 6,1013) but the instrument and reagents are rather expensive .
  • RNA detection technology A useful development in the field of RNA detection technology is the chip arrays by Affymetrix (Fodor et al , 1993 , Na ture , 364, 555; Lipshutz et al . , 1995 , BioTechniques , 19, 442) .
  • Affymetrix Fodor et al , 1993 , Na ture , 364, 555; Lipshutz et al . , 1995 , BioTechniques , 19, 442
  • thousands of different DNA oligonucleotides of known sequences can be synthesized in ordered arrays on small ( ⁇ 2cm) glass chips. These oligonucleotides can serve as capture probes to which cDNA probes generated from a particular tissue or cell type are hybridized.
  • mRNA expression levels of thousands of different genes can be monitored in a single assay.
  • This invention concerns detection and quantification of any nucleic acid sequence of interest (desired nucleic acid or target nucleic acid) from a complex pool of nucleic acid sequences.
  • the invention relates to a process for rapid simultaneous amplification of two or more RNA molecules in a large number of biological samples. More specifically, the invention relates to a process for rapid quantification of expression of a desired gene by quantifying the level of RNA in a large number of biological samples.
  • the methods disclosed in this invention also involve simultaneous amplification of a desired RNA sequence and one or more control RNA sequences, such as the house-keeping genes, in a single reaction mixture.
  • the invention concerns a process for measuring the relative amounts of two or more different nucleic acid molecules in a system.
  • the method includes: a) converting the nucleic acid molecules to tagged molecules using a 5'- and a 3'- primer specific for each nucleic acid molecule.
  • the 5' primers each have a first defined sequence
  • the 3' primers each have a second defined sequence
  • the invention features a process for measuring the relative amounts of two or more different RNA molecules in a system.
  • the invention features a process for measuring the relative amounts of two or more different DNA molecules in a system.
  • Multiplex Competitive PCR or “MC-PCR.”
  • This technique provides a means to co- amplify, in a competitive manner and in a single reaction mixture, nucleic acid molecules, for example RNA, from a single biological sample, a desired RNA sequence and RNA sequences from one or more control genes, such as the house-keeping genes mentioned above.
  • the MC-PCR strategy combines: i) the utility of using internal RNAs as controls for RNA loading and integrity that is key to Northern, RPAs and kinetic PCR strategies; ii) the precision of QC-PCR; and iii) the sensitivity of PCR amplification; all in a single reaction mixture without the need for the constant monitoring of amplification product accumulation that hampers other techniques, such as the kinetic PCR strategy.
  • the invention features a process for amplifying two or more RNA sequence by: a) cDNA synthesis (RT reaction) ; b) tagging reaction; and c) competitive amplification.
  • cDNA synthesis or the Reverse Transcription (RT) reaction as used herein, is meant, conversion of a single stranded RNA sequence into its complementary DNA (cDNA) sequence ( Figure 1A) .
  • the desired RNA sequence and control RNA sequences are converted into cDNA using enzymes, such as the reverse transcriptase .
  • the RT step is carried out using DNA oligonucleotide primers specific for each target RNA sequence. This process introduces sequences downstream from the primers, which are suitable substrates for subsequent PCR amplification.
  • tagging reaction ( Figure 1A) as used herein, is meant introducing known sequence tags at the ends of the sequence to be amplified (Reporter sequence) .
  • the Tagging reaction serves two purposes. The first purpose is to generate a second strand of DNA using the cDNA generated in the RT step as the template.
  • the second purpose of the tagging reaction is to attach defined sequences at the 5'- and 3 ' -ends of the molecule (first defined sequence and second defined sequence, respectively) .
  • defined sequences also referred to as the 5 ' -TAG sequences (first defined sequence) and 3 ' -TAG sequences (second defined sequence)
  • TAG sequence included in the 5 ' -primers is referred to as the (+)TA6 (first defined sequence), while the downstream primers (3'- primer) are referred to as (-)TAG second defined sequencer ( Figure 1A) .
  • the TAG sequences used are about 60 nt in length. Generally it is preferred that such sequences have a length between 17 and 150 bases, preferably about 40-70 bases.
  • the specific length of the TAG sequences disclosed in the example are not limiting in the invention and those skilled in the art will recognize that the length and sequence the TAG sequences can be significantly varied without significantly effecting the invention.
  • the sequences between the plus strand and negative strand TAG primers are referred to as the "reporter sequences.” These . reporter sequences are unique to each RNA being amplified. In the nonlimiting proof of principle studies described below, 24-nt regions were amplified from each mRNA to serve as the reporter sequences.
  • the specific length of the reporter sequences disclosed in the example are not limiting in the invention and those skilled in the art will recognize that the length and sequence the reporter sequences can be significantly varied without significantly effecting the invention. Generally is it preferred that such sequences have a length between 17 and 150 bases, preferably about 40-70 bases.
  • the DNA products, such as those disclosed in the non-limiting examples herein, generated after the tagging step are 180-bp long. These DNA products all contain the same (+)TAG sequence (60-bp long), followed by a 60 bp region specific for each target RNA (of which 24 bp corresponds to the reporter sequences specifically amplified by the TAG primers) , followed by the same (- )TAG (60 bp; Figure 1C) .
  • a single target RNA is co-amplified with two control house-keeping RNAs producing three different products. These three different products are identical in their 5' and 3' ends but differ in their internal regions.
  • the length of the DNA products generated after the tagging reaction can all be varied without significantly effecting the disclosed invention and hence are covered by the invention.
  • amplification primers a single pair of primers
  • generic primers specific for the (+)TAG and (-)TAG sequences
  • one generic primer has a sequence complementary to the first defined sequence ⁇ (+)TAG ⁇
  • the second generic primer has a sequence complementary to the second defined sequence ⁇ (-)TAG
  • This amplification reaction is competitive due to the use of a single primer pair to amplify the different target RNAS.
  • each of the products will accumulate to about 0.2 ⁇ M and then cease to be amplified.
  • the total amount of product is greater in multiplex reactions (reactions where 2 or more; however, each product only accumulates to about 0.2 ⁇ M.
  • This is not the case in MC-PCR because the products produced from the target and control RNAs contain a sufficient amount of common sequence -- due to the presence of the (+)TAG and (-)TAG sequences contained on all products in the reaction — that heterodimer formation is possible ( Figure IB) .
  • the critical concentration approximately 0.2 ⁇ M
  • ratios can be determined by quantifying the amount of reporter sequences present in the MC-PCR products. Differences in amounts of a target RNAs in different samples can thus be determined by comparing the ratios of the products generated by the MC-PCR. While a wide variety of techniques can be used to accomplish this, in the examples provided below, MC- PCR products were immobilized on nitrocellulose filters and hybridized with probes specific for each reporter sequence .
  • kits for use in the claimed methodology include primers and probes and enzymes as well as buffers necessary for practice of the method.
  • the kit includes a plurality of 5' and 3' primers as described above and a generic primer pair as described above.
  • the appropriate components for measurement of the relative amount of amplified tagged molecules may also be provided.
  • the enzymes that might be included include reverse transcriptase, DNA polymerase as well as the substrates for such enzymes including dNTPs, rNTPs, if desired, and appropriate labeled molecules to allow measurement of relative amount of amplified taq molecules.
  • kits may include 5' and 3' primers designed for amplification of household genes along with one or more 5' and 3' primers designed for amplification of the gene of interest.
  • One or more generic primer pairs may also be provided in such a kit as noted above.
  • Figures 1A-C Schematic representation of MC-PCR Assay.
  • A Steps involved in the MC-PCR assay.
  • B Complexes that form between multiplexed PCR products during the competitive phase of MC-PCR assay.
  • C Schematic representation of MC-PCR products generated in the following examples.
  • Figures 2A-D Comparison of Northern, RPA and MC- PCR analysis of a time course of stromelysin mRNA induction in IL-1-treated HS27 human fibroblasts.
  • A-C Northern, RPA and MC-PCR raw data are shown in Figs. 2A- C, respectively. 0, 7, 11, 15, 17 and 23, indicate hours post-induction with IL-1.
  • D Quantification of the magnitudes of stromelysin mRNA induction relative to untreated ("0"), as measured by these three different techniques .
  • FIGS 3A-C Sensitivity of MC-PCR assay. Stromelysin mRNA was induced to high levels in HS27 human fibroblasts by treatment with IL-1. Total RNA was extracted from the cells either prior to IL-1 treatment
  • FIG. 4 MC-PCR analysis of admixtures of HS27 RNA from ILl-treated ("Induced") and untreated (“Uninduced”) HS27 human fibroblasts.
  • Total mRNA from ILl-sti ulated HS27 cells ( Figure 3) was diluted with total RNA from untreated cells ( Figure 3) and analyzed by MC-PCR for Stromelysin mRNA levels.
  • FIGS 5A-B Comparison of Northern and MC-PCR analyses of serum induction of c-fos mRNA in serum- starved HeLa cells.
  • A. Time-course of c-fos mRNA induction in serum-starved HeLa cells following serum stimulation as determined by Northern analysis.
  • B. MC- PCR analysis of c-fos mRNA levels in serum-starved ( "Unstimulated” ) HeLa cells and cells stimulated with serum for 30 min. Fos mRNA levels were analyzed relative to cdc2 kinase and raf kinase mRNAs as controls.
  • amplification as used herein, is meant, production of any particular sequence from a given sequence of RNA in amounts greater than the amount initially present.
  • co-amplification is meant simultaneous amplification of more than one desired RNA sequence in a single reaction.
  • oligonucleotide as used herein, is meant a molecule comprising two or more nucleotides preferably at least 11 nucleotides, most preferably at least 17 nucleotides.
  • primers as used herein, is meant oligonucleotides comprising sequences that are complementary to a portion of the target sequence to be amplified, wherein said complementary sequence is capable of interacting with the target sequence by base- pairing interactions. This complementarity functions to allow sufficient hybridization of the primer molecule to the target sequence to allow the primer to be extended. Primer extension occurs when the primer and the target sequence are incubated under conditions suitable for primer extension in the presence of appropriate enzymes and nucleotides. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.
  • amplification primers primers comprising sequence that are complementary to either the (+)TAG sequence (first defined sequence) or the (-)TAG sequence (second defined sequence). These primers are used during the competitive amplification reaction. These primers are also referred to as ligeneric primers”.
  • nucleic acid sequence that can form hydrogen bond(s) with other nucleic acid sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-pairing interaction.
  • PCR polymerase chain reaction
  • PCR is method used to selectively amplify a desired nucleic acid sequence
  • biological sample system
  • biological sample material, in a purified or unpurified form, from biological sources, including but not limited to human, animal, plant, bacteria, viruses, fungi and the like, that contains the desired target RNA sequence (s) to be detected and/or amplified.
  • the instant invention involves detection and quantification of one or more desired RNA sequences from a complex pool of RNA sequences.
  • Purified or unpurified RNA samples can be utilized as the starting material to practice the invention.
  • the starting RNA sample may contain more than one desired target RNA sequence which may be the same or different.
  • the present invention is useful for generating large amounts of a desired RNA sequence and/or more than one desired RNA sequence, which may be same or different, located on the same or different RNA molecules .
  • the invention relates to a process for rapid high-throughput amplification of a target RNAs in a large number of biological samples. More specifically, the invention relates to a process for rapid quantification of expression of a target gene(s) by quantifying the level of mRNA in a large number of biological samples.
  • this invention involves simultaneous amplification of a desired RNA sequence and one or more control RNA sequences, such as the housekeeping genes, in a single reaction mixture.
  • Example 1 Comparison of Northern, RPA and MC-PCR techniques
  • fibroblasts normally express very low levels of the metalloprotease, stromelysin. However, fibroblasts produce large amounts of stromelysin mRNA and protein in response to IL-1 treatment. Stromelysin mRNA induction in HS27 human foreskin fibroblasts was used as a model system to demonstrate the MC-PCR assay.
  • Human foreskin fibroblasts cell line (HS-27) or primary human synovial fibroblasts (HSF) were used in this assay. All cells were plated the day before the assay in media containing 10% fetal bovine serum in 24 well plates at a density of 5xl0 4 cells/well. At 24 hours after plating, the media was removed from the wells and the monolayers were washed with Dulbeccos phosphate buffered saline (PBS) . The cells were serum starved for 24 h by incubating the cells in media containing 0.5% fetal bovine serum (FBS; 1 ml/well). Cells were washed twice with PBS containing Ca 2+ and Mg 2+ . Cells were then treated with FBS to a final concentration of 10%. Supernatants were harvested 16 hours after IL-1 induction and assayed for desired RNA expression by either RPA, Northern or MC-PCR.
  • PBS Dulbeccos phosphate buffered
  • RNA is extracted with RNeasy kits (Qiagen) , and was analyzed by Northern-blot assay. Briefly, 0.5 ⁇ g cellular RNA was separated on 1.0 % agarose/formaldehyde gel and transferred to Zeta- Probe GT nylon membrane (Bio-Rad, Hercules, CA) by capillary transfer for -16 hours. The blots were baked for two hours and then pre-hybridized for 2 hours at 65°C in 10 ml Church hybridization buffer (7 % SDS, 500 mM phosphate, 1 mM EDTA, 1 % Bovine Serum Albumin) . The blots were hybridized at 65°C for ⁇ 16 hours with 10 6 cpm/mi of full length 32 P-labeled complementary RNA
  • cRNA probes to stromelysin, ⁇ 2-microglobulin, GAPDH and other RNAs (cRNA added to the pre-hybridization buffer along with 100 ⁇ l 10 mg/ml salmon sperm DNA) .
  • the blot was rinsed once with 5% SDS, 25 mM phosphate, 1 mM EDTA and 0.5% BSA for 10 min at room temperature. This was followed by two washes (10 min each wash) with the same buffer at 65°C, which- was then followed by two washes (10 min each wash) at 65°C with 1% SDS, 25 M phosphate and 1 mM EDTA.
  • the blot was autoradiographed.
  • the blot was reprobed with a 100 nt cRNA probe to 18S rRNA as described above. Following autoradiography, the stromelysin expression was quantified by Phosphorlmaging, which is followed by normalization of the data to the 18S rRNA band intensities.
  • the assay is carried out essentially as described in the protocol provided with the Lysate Ribonuclease Protection Kit (United States Biochemical Corp.)
  • the probe for RNase protection is an RNA that is complementary to the desired RNA sequence.
  • This "antisense" probe RNA is transcribed in vi tro from a template prepared by the polymerase chain reaction in which the 5' primer was a DNA oligonucleotide containing the T7 promoter sequence.
  • the probe RNA is body labeled during transcription by including 32 P[CTP] in the reaction and purified away from unincorporated nucleotide triphosphates by chromatography on G-50 Sephadex.
  • RNA (100,000 to 250,000 cpms) is allowed to hybridize overnight at 37°C to the RNA from a cellular lysate or to RNA purified from a cell lysate. After hybridization, RNAse Ti and RNAse A are added to degrade all single-stranded RNA and the resulting products are analyzed by gel electrophoresis and autoradiography. RNAse protection analysis was carried out on cellular RNA isolated from human synovial fibroblasts that had been treated with IL-1 or untreated.
  • Oligonucleotides labeled with a #2 correspond to the primer used specifically for reverse transcription of a target mRNA.
  • Oligonucleotides labeled with a #1 are plus-strand primers used in combination with primer # 2 to convert the region of the target mRNA to be amplified during the MC-PCR reaction into double- stranded DNA. (Note that conversion of the CDNA into double-stranded DNA using primer #1 is not indicated in Figure 1A and C. This step was performed in the present studies to increase specificity of amplification of the reporter sequences. We have found that this step is not required for high-abundance mRNAs but may be necessary for low-abundance mRNAs, ie., less than 100 copies/cell) .
  • Primers #3 and #4 are nested relative to primers #1 and #2 and represent the tagging primers referred to in Figure 1A.
  • Oligonucleotides labeled #5 are the probes used to detect the reporter sequences specifically amplified by primers #3 and #4.
  • the defined sequences included in the tagging primers #3 and #4 and used during the competitive amplification step ( Figure 1A) are referred to as (+)TAG primer sequence (5'
  • cdc2 oligos cdc-1 5'- CAG ACT AGA AAG TGA AGA-3' (SEQ. ID. NO. : 1) cdc-2 5'- CCA TGT ACT GAC CAG GAG-3 ' (SEQ. ID. NO. : 2) cdc-3 5'- ACA TTC TCA AGT CTG ACT TTG CCT TTG CAT AAC AAG CAC TTA GCA TTA ACC CTC ACT AAA CGT CAT CCA AAT ATA GTC-3'
  • K-ras oligos ras-1 5'- TAC AGC TAA TTC AGA ATC-3' (SEQ. ID. NO.: 6) ras-2 5'- CAA GAG ACA GGT TTC TCC-3' (SEQ. ID. NO.: 7) ras-3 5'- ACA TTC TCA AGT CTG ACT TTG CCT TTG CAT AAC AAG CAC TTA GCA TTA ACC CTC ACT AAA ATT TTG TGG ACG AAT ATG-3'
  • C-fos oligos fos-1 5'- TAA GAT GGC TGC AGC CGA-3' (SEQ. ID. NO. : 11) fos-2 5'- CAG TTT GGC AAT CTC TGT CTG-3'
  • c-raf oligos raf-1 5'- TCG TAT GCG AGA GTC TGT-3' (SEQ. ID. NO.: 16) raf-2 5*- CCT TCA GAT GAG GGA CTG-3' (SEQ. ID. NO.: 17) raf-3 5'- ACA TTC TCA AGT CTG ACT TTG CCT TTG CAT AAC AAG CAC TTA GCA TTA ACC CTC
  • RNA purification was purified using the RNeasy kits (Qiagen) .
  • RNA Reverse transcription and conversion to double stranded DNA.
  • Sample RNA ranging from 10 ng to 1 ⁇ g in 5 ⁇ l of water, was combined in thin-walled 0.5ml Eppendorf microcentrifuge tubes with 1 ⁇ l of a RT primer mix consisting of 5 ⁇ M of primer #2 for each target to be analyzed. The resulting mixtures were heated to 90EC for 3 minutes, then slow-cooled on the bench top to promote hybridization of primers #2 to the target RNA.
  • Reverse transcription was accomplished by adding 20 ml of RT Mix consisting of 1.36X PCR reaction buffer
  • Tagging step The defined (+)TAG and (-)TAG sequences were incorporated onto the ends of the reporter sequences of each target RNA using primers #3 and #4 by addition of 75 ⁇ l of Tag Mix containing 0.2 ⁇ M each of primers #3 and #4 for each target RNA, IX Taq buffer and 0.2 mM dNTPs, to the DNA reactions and subjecting the reactions to 5 cycles of (94°C for 30 s, 45°C for 45 s and 72°C for 1 min) . Reactions were then held at 72°C following the final cycle until retrieved from the thermocycler to inhibit production of primer dimers between the complex mixture of tagging primers, then placed on ice. Multiplexed competitive amplification step.
  • the resulting doublestranded DNA products from each of the target RNAs were then amplified with a common set of TAG primers by transferring 2 ⁇ l from the tagging reaction to 100 ⁇ l of MC reaction mix consisting of lx Taq buffer, 0.2 mM dNTPs, 0.2 ⁇ M each of TAG primers and 5 U Amplitaq Gold, heating the reactions to 9.4°C for 10 min to activate the AmpliTaq Gold DNA polymerase and subjecting the reactions to 50 cycles of (94°C for 30 s, 60°C for 30 s and 72°C for 1 min) . Aliquots (5 ⁇ l) from each reaction were analyzed on 4% agarose gels to insure that appropriate-sized product (180 bp) was produced.
  • the relative amounts of product produced from each of the target RNAs during the multiplexed competitive amplification step were quantified in the present studies by filter hybridization using probes specific for the reporter sequences amplified.
  • 10 ⁇ l of product was combined with 1 ul of 2N NaOH/0.2 M EDTA in 1.5 ml Eppendorf centrifuge tubes and heated to 37°C for 30 min to denature the product (thus, if 3 targets were multiplexed, then 30 ⁇ l of product would be combined with 3 ml of sodium hydroxide solution) .
  • the tubes were then chilled on ice and 230 ml of ice cold 20x SSC was added per target RNA analyzed.
  • FIG. 2 it compares Northern, RPA and MC-PCR analyses of a time course of stromelysin mRNA induction following IL-1 treatment.
  • Total RNA was harvested from the HS27 cells at different time points following IL-1 addition to the growth medium.
  • Stromelysin mRNA was analyzed either by Northern analysis using 5 ⁇ g total RNA (Figure 2A) , RPA analysis of 2 ⁇ g total RNA ( Figure 2B) , or MC-PCR analysis of 1 ⁇ g total RNA ( Figure 1C) .
  • GAPDH mRNA was used as the house-keeping control gene in the Northern and RPA assays to normalize for loading differences and for RNA integrity.
  • stromelysin mRNA levels were calculated in Northern and RPA studies by normalizing the hybridization signals of a probe to stromelysin RNA relative to hybridization signals of a probe to GAPDH RNA followed by the plotting of the normalized signals relative to levels observed in RNA from untreated HS27 cells.
  • relative induction of stromelysin mRNA was calculated using the above formula.
  • IL-1 induction of stromelysin mRNA in HS27 cells was used to address the sensitivity of the MC-PCR technique.
  • FIG. 3A Figure 3A and 1 ⁇ g by MC-PCR ( Figure 3B) .
  • ⁇ 2-microglobulin was used as the control probe in the Northern analysis ( Figure 3A) .
  • the Northern analysis indicated that stromelysin mRNA was induced about 30-fold; however, the stromelysin signal was difficult to quantify due to the low signal over background (see Figure 3A lane "Uninduced") which greatly affects the accuracy of measuring the magnitude of stromelysin mRNA induction. This problem was not encountered in the MC-PCR assay due to the inclusion of the PCR amplification step which amplified the stromelysin signal in the uninduced RNA sample well above noise (see Figure. 3B) .
  • RNA samples were used to test the sensitivity of the MC-PCR assay. Varying amounts of RNA, ranging from 1 mg down to 0.01 ⁇ g, from either uninduced or IL-1 -stimulated HS27 cells was analyzed in triplicate as in Figure 3B and the hybridization signals were quantified and graphed ( Figure 3C) . Similar ratios of GAPDH, ⁇ 2-microglobulin and stromelysin amplification products were obtained from either the untreated or IL-1 stimulated samples regardless of the amount of total RNA analyzed.
  • RNA as little as 10 ng of total RNA, equivalent to the amount of RNA obtained from 1000 cells, was sufficient to obtain very reproducible results with MC-PCR (error bars represent standard deviations) .
  • the amount of RNA required for Northern analysis is 5 ⁇ g which is 500 times greater than the lowest amount of RNA tested in Figure 3C.
  • Example 3 Precision of MC-PCR reaction The MC-PCR assay was sufficiently robust to detect differences in the induction levels of stromelysin mRNA in the above examples, which ranged from about 7-fold
  • MC-PCR assay to be used to discriminate smaller changes in stromelysin RNA was tested by diluting total RNA extracted from the IL-1 -stimulated HS27 cells used in Figure 3 with RNA from the untreated sample and measuring changes in stromelysin mRNA in the resulting admixtures ( Figure 4). Changes in stromelysin mRNA levels were readily detected in all of the admixtures indicating the ability of the assay to identify small differences (less than 2-fold) in mRNA abundances.
  • Example 4 MC-PCR analysis of low-copy number mRNAs.
  • GAPDH GAPDH
  • ⁇ 2-microglobulin and stromelysin mRNAs represent relatively abundant mRNAs (greater than 1000 copies per cell) in IL-1 -stimulated HS27 cells.
  • MC-PCR method we compared results obtained from Northern and MC-PCR analyses of serum induction of c-fos mRNA in HeLa cells, c-fos mRNA is extremely low in serum-starved HeLa cells, but is induced more than 20-fold 30 minutes after addition of serum (Figure 5A) .
  • Example 5 MC-PCR analysis of antisense inhibition of cdc2 kinase mRNA
  • HeLa cells were treated for 4 hours with either a phosphorothioate antisense oligonucleotide targeting cdc2 kinase mRNA or a mismatch control, complexed at a 4:1 charge ratio with Pfx4 (Life Technologies) in OptiMEM ® (BRL/Gibco) . Cells were then washed, cultured for an additional 20 hours in complete media, RNA was harvested and 400 ng was analyzed by MC- PCR using oligonucleotides specific for cdc2 kinase, and K-ras (ras) as a control target RNA. Error bars represent standard deviations.
  • the present invention can be used to quantify changes in abundance of target DNA or RNA sequences in response to any external or environmental stimuli including but not limited to antisense, ribozymes or drug treatment can be readily accomplished using the instant invention. Quantifying changes in abundance of target DNA or
  • RNA sequences during development or differentiation of a cell type or tissue, or during development or abbrogation of a particular disease phenotype can be readily accomplished using the instant invention.
  • the instant invention can be used to detect the presence of, and/or quantify the relative amounts of, infectious agents including but not limited to viruses, bacteria, fungi, protazoa or the like in biological samples for diagnostic or prognostic purposes.
  • Detecting or quantifying presence of exogenous DNA or RNA sequences including but not limited to vectors or the like used for the purposes of gene therapy.

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  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de mesure des quantités relatives de deux molécules d'acides nucléiques différentes ou davantage dans un système biologique au moyen d'une amplification en chaîne par polymérase multiplex compétitive.
PCT/US1998/001471 1997-02-07 1998-01-27 Procede ameliore de detection et de quantification de molecules d'acides nucleiques WO1998035058A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63173/98A AU6317398A (en) 1997-02-07 1998-01-27 Improved process for detection and quantification of nucleic acid molecules

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US3784197P 1997-02-07 1997-02-07
US60/037,841 1997-02-07
US99373197A 1997-12-18 1997-12-18
US08/993,731 1997-12-18

Publications (2)

Publication Number Publication Date
WO1998035058A2 true WO1998035058A2 (fr) 1998-08-13
WO1998035058A3 WO1998035058A3 (fr) 1998-09-17

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PCT/US1998/001471 WO1998035058A2 (fr) 1997-02-07 1998-01-27 Procede ameliore de detection et de quantification de molecules d'acides nucleiques

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WO (1) WO1998035058A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002048352A1 (fr) * 2000-12-12 2002-06-20 Aisin Seiki Kabushiki Kaisha Methode d'analyse d'expression genique
WO2003060159A3 (fr) * 2002-01-15 2004-01-22 Matforsk Techniques d'amplification d'acide nucleique
EP1194592A4 (fr) * 1999-07-14 2004-12-08 Packard Bioscience Company Acides nucleiques modifies et utilisations associees
US6964847B1 (en) 1999-07-14 2005-11-15 Packard Biosciences Company Derivative nucleic acids and uses thereof
WO2005116248A1 (fr) * 2004-05-26 2005-12-08 Jakob Stenman Methode de mesure quantitative et/ou comparative des niveaux d'expression de l'arnm dans de petits echantillons biologiques
WO2006033928A3 (fr) * 2004-09-16 2007-03-15 Applera Corp Compositions, methodes et trousses permettant d'identifier et de quantifier des petites molecules d'arn
US9068222B2 (en) 2004-05-28 2015-06-30 Applied Biosystems, Llc Methods compositions, and kits comprising linker probes for quantifying polynucleotides

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7125660B2 (en) 2000-09-13 2006-10-24 Archemix Corp. Nucleic acid sensor molecules and methods of using same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5643765A (en) * 1993-04-06 1997-07-01 University Of Rochester Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction
US5422252A (en) * 1993-06-04 1995-06-06 Becton, Dickinson And Company Simultaneous amplification of multiple targets

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1194592A4 (fr) * 1999-07-14 2004-12-08 Packard Bioscience Company Acides nucleiques modifies et utilisations associees
US6964847B1 (en) 1999-07-14 2005-11-15 Packard Biosciences Company Derivative nucleic acids and uses thereof
WO2002048352A1 (fr) * 2000-12-12 2002-06-20 Aisin Seiki Kabushiki Kaisha Methode d'analyse d'expression genique
WO2003060159A3 (fr) * 2002-01-15 2004-01-22 Matforsk Techniques d'amplification d'acide nucleique
WO2005116248A1 (fr) * 2004-05-26 2005-12-08 Jakob Stenman Methode de mesure quantitative et/ou comparative des niveaux d'expression de l'arnm dans de petits echantillons biologiques
US7838228B2 (en) 2004-05-26 2010-11-23 Expression Analytics Oy Method of quantitative and/or comparative measurement of mRNA expression levels in small biological samples
US9068222B2 (en) 2004-05-28 2015-06-30 Applied Biosystems, Llc Methods compositions, and kits comprising linker probes for quantifying polynucleotides
US9657346B2 (en) 2004-05-28 2017-05-23 Applied Biosystems, Llc Methods, compositions, and kits comprising linker probes for quantifying polynucleotides
US10781486B2 (en) 2004-05-28 2020-09-22 Applied Biosystems, Llc Methods, compositions, and kits comprising linker probes for quantifying polynucleotides
US11891663B2 (en) 2004-05-28 2024-02-06 Applied Biosystems, Llc Methods, compositions, and kits comprising linker probes for quantifying polynucleotides
WO2006033928A3 (fr) * 2004-09-16 2007-03-15 Applera Corp Compositions, methodes et trousses permettant d'identifier et de quantifier des petites molecules d'arn

Also Published As

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