Abstract
The reverse-transcriptase-polymerase chain reaction (RT-PCR) can be used to determine minute amounts of mRNAs in tissue samples by co-amplification of a quantified amount of a competitive sequence (internal standard), so-called quantitative competitive (qc) RT-PCR. The first description of qcRT-PCR was provided by Wang et al. (1). As an internal standard, an RNA template with the same primer sites as the target mRNA was reverse-transcribed. The amplified PCR products differed in size and were separated by agarose gel electrophoresis. The presence of 32P-labeled 5′ primer allowed quantification by scintillation counting.
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References
Wang A.M., Doyle M.V., and Mark D.F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 9717–9721.
Diviacco S., Norio P., Zentilin L., Menzo S., Clementi M., Biamont G., et al. (1992) A novel procedure for quantitative polymerase chain reaction coamplification of competitive templates. Gene 122, 313–320.
Gilliland G., Perrin S., Blanchard K., and Bunn H.F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 2725–2729.
Zhou N.-M., Matthys P., Polacek C., Fiten P., Sato A., Billiau A., et al. (1997) A competitive RT-PCR Method for the Quantitative Analysis of Cytokine mRNAs in Mouse Tissues. Cytokine 9, 212–218.
Hess J.F., Borkowski J.A., Young G.S., Strader C.D., and Ransom R.W. (1992) Cloning and pharmacological characterization of a human bradykinin (BK-2)receptor. Biochem. Biophys. Res. Commun. 184, 260–268.
de Breuil R.M., Patel J.M., and Mendelow B.V. (1993) Quantification of betaactin-specific mRNA transcripts using xeno-competitive PCR. PCR Methods Appl. 3, 57–59.
Ulfelder K.J., Schwartz H.E., Hall J.M., and Sunzeri F.J. (1992) Restriction fragment length polymorphism analysis of ERB B2 oncogene by capillary electrophoresis. Anal. Biochem. 200, 260–267.
Murata T., Takizawa T., Funaba M., Fujimura H., Murata A., and Torii K. (1997) Quantitation of mouse and rat β-actin mRNA by competitive polymerase chain reaction using capillary electrophoresis. Anal. Biochem. 244, 172–174.
Watson D.K., McWilliams M.J., Lapis P., Lautenberger J.A., Schweinfest C.W., and Papas T.S. (1988) Mammalian ets-1 and ets-2 genes encode highly conserved proteins. Proc. Natl. Acad. Sci. USA 85, 7862–7866.
Vandekerckhove J., and Weber K. (1978) Mammalian cytoplasmic actins are the products of at least two genes and differ in primary structure in at least 25 identified positions from skeletal muscle actins. Proc. Natl. Acad. Sci. USA 75, 1106–1110.
VanStraaten F., Müller R., Curran T., BanBeveren C., and Verma I.M. (1983) Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein. Proc. Natl. Acad. Sci. USA 80, 3183–3187.
Zhao N., Hashida H., Takahashi N., and Sakaki Y. (1994) Cloning and sequence analysis of the human snap 25 cDNA. Gene 145, 313–314.
Rychlik W., and Rhoads R.E. (1989) A computer program for choosing optimal oligonucleotide primers for filter hypbridization, sequencing and in vitro amplification. Nucleic Acids Res. 17, 8543–8551.
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© 2002 Humana Press Inc.
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Greber-Platzer, S., Balcz, B., Fleischmann, C., Lubec, G. (2002). Using the Quantitative Competitive RT-PCR Technique to Analyze Minute Amounts of Different mRNAs in Small Tissue Samples. 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:029
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DOI: https://doi.org/10.1385/1-59259-283-X:029
Publisher Name: Humana Press
Print ISBN: 978-0-89603-875-2
Online ISBN: 978-1-59259-283-8
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