Abstract
The powerful amplification potential of PCR has assured its use in the detection of low-abundance mRNA in cells and tissues. RT-PCR is currently the most sensitive technique available for mRNA detection and quantification. It can accurately quantify genes present at only a few hundred copies per sample, and has become the method of choice for the examination of gene expression. The technique consists of two parts: synthesis of cDNA from RNA by reverse transcription, and amplification of a specific cDNA by polymerase chain reaction (PCR). The method requires very little RNA and differs from Northern blotting because it is somewhat tolerant of degraded RNA, as long as the RNA is intact within the region of interest.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mansur N., Meyer-Siegler K., Wurzer J., and Sirover M. (1993) Cell cycle regulation of the glyceraldehyde-2-phosphate dehydrogenase/Uracil DNA glycosylase gene in normal human cells. Nucleic Acids Rese. 4, 993–998
Finnegan M., Goepel J., Hancock B., and Goyns M. (1993) Investigation of the expression of housekeeping genes in Non-Hodgkin’s Lymphoma. Leuk. Lymphoma 10, 387–393.
Chang T., Juan C., Yin P., Chi C., and Tsay H. (1998) Upregulation of Betaactin, cyclophilin and GAPDH in NISI Rat Hepatoma. Oncol. Rep. 5, 469–471
Foss D., Baarsch M., and Murtaugh M. (1998) Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phospate dehydrogenase and Betaactin mRNA expression in porcine immune cells and tissues. Anim. Biotechnol. 9, 67–78
Labuhn M., and Brack C (1997) Age-related changes in mRNA expression of actin isoforms in Drosophilia melangaster. Gerontology 43, 261–267
Raff T. (1997) Design and testing of β-actin primers for RT-PCR which do not co-amplify processed pseudogenes. Biotechniques 23, 456–460
O’Connell J., Goode T., and Shanahan F. (1992) Quantitative measurement of mRNA expression by competitive RT-PCR, in PCR in Bioanalysis, Meltzer S.J., ed. Humana Press, Inc., Totowa, NJ, pp. 183–195.
Überla K., Platzer C., Diamantstein T., and Blankenstein T. (1991) Generation of competitor DNA fragments for quantitative PCR. PCR Methods Appl. 1, 136–139.
Siebert P.D., and Larrick J.W. (1993) PCR MIMICs: competitive DNA fragments for use as internal standards in quantitative PCR. Biotechniques 14, 244–249
Siebert P.D. (1999) Quantitaitve RT-PCR, in Quantitative PCR Protocols, Kochanowski B., and Udo Reischl, eds., Humana, Totowa, NJ, pp. 61–85.
Celi F.S., Zenilman M.E., and Shuldiner A.R. (1993) A Rapid and versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Research 21(4), 1047.
Porcher C., Malinge M.C., Picat C., and Grandchamp B. (1992) A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer. Biotechniques 13, 106–113
Overbergh L., Valckx D., Waer D., and Mathieu C. (1999) Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11(4), 305–312
Halminen M., Sjoroois M., Makela M.J., Waris M., Terho E., Lovgren T., et al. (1999) Simultaneous detection of IFN-[g] and IL-4 mRNAs using RT-PCR and time-resolved fluorometry. Cytokine 11(1), 87–93
Alard P., Lantz O., Sebagh M., Calvo C.F., Weill D., Chavanel G., et al. (1993) A versatile ELISA-PCR assay for mRNA quantification from a few cells. Biotechniques 15, 730–737
Dufour V., Arnauld C., Lantz O., Peguillet I., Morvilliers K., Emmanuel A., et al. (1999) Quantification of porcine cytokine gene expression using RT-PCR, a homologous internal control and chemiluminescence for microplate detection. J. Immunol. Methods 229, 49–60
Motmans K., Raus J., and Vandevyver C. (1996) Quantification of cytokine messenger RNA in transfected human T cells by RT-PCR and an automated electrochemiluminescence-based post-PCR detection system. J. Immunol. Methods 190, 107–116
Zimmermann K., and Mannhalter J. W. (1996) Technical aspects of Quantitative PCR. Biotechniques 21, 268–279.
Bouaboula M., Legoux P., Pességué B., Dumont X., Piechaczyk M., Casellas P., et al. (1992) Standardisation of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control. J. Biol. Chem. 267, 21,830–21,838
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Joyce, C. (2002). Quantitative RT-PCR. In: O’Connell, J. (eds) RT-PCR Protocols. Methods in Molecular Biology, vol 193. Humana Press. https://doi.org/10.1385/1-59259-283-X:083
Download citation
DOI: https://doi.org/10.1385/1-59259-283-X:083
Publisher Name: Humana Press
Print ISBN: 978-0-89603-875-2
Online ISBN: 978-1-59259-283-8
eBook Packages: Springer Protocols