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. 1998 Jun 1;26(11):2511–2518. doi: 10.1093/nar/26.11.2511

Modeling and analysis of competitive RT-PCR.

A L Hayward 1, P J Oefner 1, S Sabatini 1, D B Kainer 1, C A Hinojos 1, P A Doris 1
PMCID: PMC147587  PMID: 9592131

Abstract

The present studies demonstrate a theoretical and practical framework for the accurate quantitation of gene expression in RNA extracted from microscopic tissue samples. The approaches are developed around competitive RT-PCR techniques. Assay performance has been examined and validated at both the RT and PCR steps. Our analysis of RT transcription efficiency for a number of native and competitor combinations shows that this property can differ, even for very similar templates. However, this difference is consistent and, once identified and measured, can be removed as an obstacle to accuracy. Using mathematical modeling, we have examined the simulated co-amplification of native and competitor templates in PCR. Useful insights have emerged from such modeling which indicate that differences in initial amplification efficiency and the rate of decay of amplification efficiency during the reaction can rapidly lead to inaccuracy, even while the slope and linearity of log plots of the competitor input and reaction product ratios are close to ideal. Finally, we show here that competitive RT-PCR reactions do not have to remain in the log-linear phase of PCR in order to accomplish accurate and precise quantification. Using appropriate competitors sharing primer binding sites and high internal sequence similarity, identical amplification efficiencies are preserved throughout the reaction. Reaction products, including heteroduplexes formed between native and competitor templates as reactions progress to plateau, can be identified and quantified accurately using the new technique of denaturing HPLC (dHPLC). This analytical technique allows the accuracy of competitive RT-PCR to be preserved beyond the linear phase. The technique has high sensitivity and precision and target abundances as low as 100 copies could be reliably estimated.

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Selected References

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

  1. Becker-André M., Hahlbrock K. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 1989 Nov 25;17(22):9437–9446. doi: 10.1093/nar/17.22.9437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ferre F. Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 1992 Aug;2(1):1–9. doi: 10.1101/gr.2.1.1. [DOI] [PubMed] [Google Scholar]
  3. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hayward-Lester A., Oefner P. J., Doris P. A. Rapid quantification of gene expression by competitive RT-PCR and ion-pair reversed-phase HPLC. Biotechniques. 1996 Feb;20(2):250–257. doi: 10.2144/96202rr02. [DOI] [PubMed] [Google Scholar]
  5. He Q., Marjamäki M., Soini H., Mertsola J., Viljanen M. K. Primers are decisive for sensitivity of PCR. Biotechniques. 1994 Jul;17(1):82, 84, 86-7. [PubMed] [Google Scholar]
  6. Henley W. N., Schuebel K. E., Nielsen D. A. Limitations imposed by heteroduplex formation on quantitative RT-PCR. Biochem Biophys Res Commun. 1996 Sep 4;226(1):113–117. doi: 10.1006/bbrc.1996.1319. [DOI] [PubMed] [Google Scholar]
  7. Huber C. G., Oefner P. J., Preuss E., Bonn G. K. High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles. Nucleic Acids Res. 1993 Mar 11;21(5):1061–1066. doi: 10.1093/nar/21.5.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ito A., Hashimoto T., Hirai M., Takeda T., Shuntoh H., Kuno T., Tanaka C. The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A. Biochem Biophys Res Commun. 1989 Sep 29;163(3):1492–1497. doi: 10.1016/0006-291x(89)91148-0. [DOI] [PubMed] [Google Scholar]
  9. Iwai N., Inagami T. Molecular cloning of a complementary DNA to rat cyclophilin-like protein mRNA. Kidney Int. 1990 Jun;37(6):1460–1465. doi: 10.1038/ki.1990.136. [DOI] [PubMed] [Google Scholar]
  10. Melo J. V., Yan X. H., Diamond J., Lin F., Cross N. C., Goldman J. M. Reverse transcription/polymerase chain reaction (RT/PCR) amplification of very small numbers of transcripts: the risk in misinterpreting negative results. Leukemia. 1996 Jul;10(7):1217–1221. [PubMed] [Google Scholar]
  11. Pallansch L., Beswick H., Talian J., Zelenka P. Use of an RNA folding algorithm to choose regions for amplification by the polymerase chain reaction. Anal Biochem. 1990 Feb 15;185(1):57–62. doi: 10.1016/0003-2697(90)90254-7. [DOI] [PubMed] [Google Scholar]
  12. Peccoud J., Jacob C. Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys J. 1996 Jul;71(1):101–108. doi: 10.1016/S0006-3495(96)79205-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Raeymaekers L. Quantitative PCR: theoretical considerations with practical implications. Anal Biochem. 1993 Nov 1;214(2):582–585. doi: 10.1006/abio.1993.1542. [DOI] [PubMed] [Google Scholar]
  14. Wiesner R. J., Beinbrech B., Rüegg J. C. Quantitative PCR. Nature. 1993 Dec 2;366(6454):416–416. doi: 10.1038/366416b0. [DOI] [PubMed] [Google Scholar]
  15. Zuker M. Prediction of RNA secondary structure by energy minimization. Methods Mol Biol. 1994;25:267–294. doi: 10.1385/0-89603-276-0:267. [DOI] [PubMed] [Google Scholar]

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