Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717

Quantitation of mRNA by the polymerase chain reaction.

A M Wang 1, M V Doyle 1, D F Mark 1
PMCID: PMC298572  PMID: 2481313

Abstract

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.

Full text

PDF
9719

Images in this article

9719Image
on p.9719
9720Image
on p.9720

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almoguera C., Shibata D., Forrester K., Martin J., Arnheim N., Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 1988 May 20;53(4):549–554. doi: 10.1016/0092-8674(88)90571-5. [DOI] [PubMed] [Google Scholar]
  2. Barrett T. B., Benditt E. P. Platelet-derived growth factor gene expression in human atherosclerotic plaques and normal artery wall. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2810–2814. doi: 10.1073/pnas.85.8.2810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrett T. B., Benditt E. P. sis (platelet-derived growth factor B chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1099–1103. doi: 10.1073/pnas.84.4.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Betsholtz C., Johnsson A., Heldin C. H., Westermark B., Lind P., Urdea M. S., Eddy R., Shows T. B., Philpott K., Mellor A. L. cDNA sequence and chromosomal localization of human platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature. 1986 Apr 24;320(6064):695–699. doi: 10.1038/320695a0. [DOI] [PubMed] [Google Scholar]
  5. Chelly J., Kaplan J. C., Maire P., Gautron S., Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature. 1988 Jun 30;333(6176):858–860. doi: 10.1038/333858a0. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Claesson-Welsh L., Eriksson A., Morén A., Severinsson L., Ek B., Ostman A., Betsholtz C., Heldin C. H. cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules. Mol Cell Biol. 1988 Aug;8(8):3476–3486. doi: 10.1128/mcb.8.8.3476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Collins T., Ginsburg D., Boss J. M., Orkin S. H., Pober J. S. Cultured human endothelial cells express platelet-derived growth factor B chain: cDNA cloning and structural analysis. Nature. 1985 Aug 22;316(6030):748–750. doi: 10.1038/316748a0. [DOI] [PubMed] [Google Scholar]
  9. Fujita T., Takaoka C., Matsui H., Taniguchi T. Structure of the human interleukin 2 gene. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7437–7441. doi: 10.1073/pnas.80.24.7437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  11. Horn G. T., Bugawan T. L., Long C. M., Erlich H. A. Allelic sequence variation of the HLA-DQ loci: relationship to serology and to insulin-dependent diabetes susceptibility. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6012–6016. doi: 10.1073/pnas.85.16.6012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kashani-Sabet M., Rossi J. J., Lu Y., Ma J. X., Chen J., Miyachi H., Scanlon K. J. Detection of drug resistance in human tumors by in vitro enzymatic amplification. Cancer Res. 1988 Oct 15;48(20):5775–5778. [PubMed] [Google Scholar]
  13. Kawasaki E. S., Clark S. S., Coyne M. Y., Smith S. D., Champlin R., Witte O. N., McCormick F. P. Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5698–5702. doi: 10.1073/pnas.85.15.5698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kawasaki E. S., Ladner M. B., Wang A. M., Van Arsdell J., Warren M. K., Coyne M. Y., Schweickart V. L., Lee M. T., Wilson K. J., Boosman A. Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1). Science. 1985 Oct 18;230(4723):291–296. doi: 10.1126/science.2996129. [DOI] [PubMed] [Google Scholar]
  15. Larzul D., Guigue F., Sninsky J. J., Mack D. H., Bréchot C., Guesdon J. L. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J Virol Methods. 1988 Jul;20(3):227–237. doi: 10.1016/0166-0934(88)90126-7. [DOI] [PubMed] [Google Scholar]
  16. Luskey K. L., Stevens B. Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation. J Biol Chem. 1985 Aug 25;260(18):10271–10277. [PubMed] [Google Scholar]
  17. March C. J., Mosley B., Larsen A., Cerretti D. P., Braedt G., Price V., Gillis S., Henney C. S., Kronheim S. R., Grabstein K. Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs. Nature. 1985 Jun 20;315(6021):641–647. doi: 10.1038/315641a0. [DOI] [PubMed] [Google Scholar]
  18. Myerowitz R. Splice junction mutation in some Ashkenazi Jews with Tay-Sachs disease: evidence against a single defect within this ethnic group. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3955–3959. doi: 10.1073/pnas.85.11.3955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nedwin G. E., Naylor S. L., Sakaguchi A. Y., Smith D., Jarrett-Nedwin J., Pennica D., Goeddel D. V., Gray P. W. Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Res. 1985 Sep 11;13(17):6361–6373. doi: 10.1093/nar/13.17.6361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Neri A., Knowles D. M., Greco A., McCormick F., Dalla-Favera R. Analysis of RAS oncogene mutations in human lymphoid malignancies. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9268–9272. doi: 10.1073/pnas.85.23.9268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Paik Y. K., Chang D. J., Reardon C. A., Davies G. E., Mahley R. W., Taylor J. M. Nucleotide sequence and structure of the human apolipoprotein E gene. Proc Natl Acad Sci U S A. 1985 May;82(10):3445–3449. doi: 10.1073/pnas.82.10.3445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rappolee D. A., Mark D., Banda M. J., Werb Z. Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science. 1988 Aug 5;241(4866):708–712. doi: 10.1126/science.3041594. [DOI] [PubMed] [Google Scholar]
  23. Rappolee D. A., Wang A., Mark D., Werb Z. Novel method for studying mRNA phenotypes in single or small numbers of cells. J Cell Biochem. 1989 Jan;39(1):1–11. doi: 10.1002/jcb.240390102. [DOI] [PubMed] [Google Scholar]
  24. Smith M. F., Jr, Kueppers F. R., Young P. R., Lee J. C. A rapid and quantitative method for the determination of interleukin-1 alpha and -beta mRNA expression in human monocytes and macrophages. J Immunol Methods. 1989 Mar 31;118(2):265–272. doi: 10.1016/0022-1759(89)90015-x. [DOI] [PubMed] [Google Scholar]
  25. Todd J. A., Bell J. I., McDevitt H. O. HLA-DQ beta gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature. 1987 Oct 15;329(6140):599–604. doi: 10.1038/329599a0. [DOI] [PubMed] [Google Scholar]
  26. Wion K. L., Kirchgessner T. G., Lusis A. J., Schotz M. C., Lawn R. M. Human lipoprotein lipase complementary DNA sequence. Science. 1987 Mar 27;235(4796):1638–1641. doi: 10.1126/science.3823907. [DOI] [PubMed] [Google Scholar]
  27. Wu D. Y., Ugozzoli L., Pal B. K., Wallace R. B. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2757–2760. doi: 10.1073/pnas.86.8.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yamamoto T., Davis C. G., Brown M. S., Schneider W. J., Casey M. L., Goldstein J. L., Russell D. W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell. 1984 Nov;39(1):27–38. doi: 10.1016/0092-8674(84)90188-0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES