Quantification of Endothelin Receptor mRNA by Competitive RT-PCR
Part of the
Methods in Molecular Biology™
book series (MIMB, volume 206)
The potent effects of the endothelins (ETs), including their vasoconstrictor, positive inotropic and co-mitogenic actions, are mediated by at least two distinct ET receptor subtypes, ETA (1) and ETB (2). The ETA receptor is selective for ET-1, with binding affinity ET-1>ET-2≫ET-3, while the ETB receptor is nonisoform selective. Both subtypes are structurally similar, having seven transmembrane domains characteristic of the G-protein-coupled superfamily (1,2). In several pathophysiological conditions, including myocardial infarction (3), congestive heart failure (4), and renal failure (5), there is altered expression of ET receptors. These changes further implicate ET in the pathogenesis of such conditions and provide additional characterization of the disease process. It is, therefore, essential to have an accurate and reliable means of measuring ET receptor expression. Traditional Northern analysis has the disadvantage of low sensitivity, and while the reverse transcription-polymerase chain reaction (RT-PCR) offers 1000–10,000-fold greater sensitivity, the exponential nature of its amplification kinetics makes it difficult to obtain truly quantitative information. Competitive RT-PCR obviates this problem by co-amplifying the gene of interest with a known concentration of mutant cDNA, which as the name suggests, competes for primer binding and PCR substrates. The subsequent PCR products from wild-type and mutant are distinguished by size or the presence or absence of a restriction enzyme site. By constructing plots of the ratio of wild-type to competitor densities vs molar concentration of competitor cRNA, the starting concentration of the wild-type RNA can be calculated.
KeywordsLeukemia Agarose Bromide Electrophoresis MgCl2
Arai H., Hori S., Aramori I., Ohkubo H., and Nakanishi S. (1990) Cloning and expression of a cDNA encoding an endothelin receptor. Nature
, 730–732.PubMedCrossRefGoogle Scholar
Sakurai T., Yanagisawa M., Takuwa Y., Miyazaki H., Kimura S., Goto K., and Masaki T. (1990) Cloning of a cDNA encoding a nonisopeptide-selective subtype of the endothelin receptor. Nature
, 732–735.PubMedCrossRefGoogle Scholar
Nambi P., Pullen M., Egan J. W., and Smith E. F. (1991) Identification of cardiac endothelin binding sites in rats: downregulation of left atrial endothelin binding sites in response to myocardial infarction. Pharmacology
, 84–89.CrossRefGoogle Scholar
Morawietz H., Szibor M., Goettsch W., Bartling B., Barton M., Shaw S., et al. (2000) Deloading of the left ventricle by ventricular assist device normalizes increased expression of entothelin ET(A) receptors but not endothelin-converting enzyme-1 in patients with end-stage heart failure. Circulation
102(19 Suppl. 3)
, III188–193.PubMedGoogle Scholar
Shimizu T., Hata S., Kuroda T., Mihara S., and Fujimoto M. (1999) Different roles of two types of endothelin receptors in partial ablation-induced chronic renal failure in rats. Eur. J. Pharmacol.
, 39–49.PubMedCrossRefGoogle Scholar
Smith P. J., Brooks J. I., Stewart D. J., and Monge J. C. (1999) Quantification of endothelin ETA and ETB receptor mRNA by competitive reverse transcription-polymerase chain reaction: development of amultispecies assay. Anal. Biochem.
, 93–96.PubMedCrossRefGoogle Scholar
Picard P., Smith P. J. W., Monge J. C., Rouleau J. L., Nguyen Q. T., Calderone A., and Stewart D. J. (1998) Coordinated upregulation of the cardiac endothelin system in a rat model of heart failure. J. Cardiovasc. Pharmacol.
, S294–S297.CrossRefGoogle Scholar
Smith P. J. W., Ornatsky O., Stewart D. J., Picard P., Dawood F., Wen W. H., et al. (2000) Effects of estrogen replacement on infarct size, cardiac remodeling, and the endothelin system after myocardial infarction in ovariectomized rats. Circulation
, 2983–2989.PubMedGoogle Scholar
Picard P., Smith P. J. W., Monge J. C., and Stewart D. J. (1998) Expression of endothelial factors after arterial injury in the rat. J. Cardiovasc. Pharmacol.
, S323–S327.PubMedCrossRefGoogle Scholar
Altschul S. F., Gish W., Miller W., Myers E. W., and Lipman D. J. (1990) Basic local alignment search tool. J. Mol. Biol.
, 403–410.PubMedGoogle Scholar
Sarkar G. and Sommer S. S. (1990) The “megaprimer” method of site-directed mutagenesis. BioTechniques
, 404–407.PubMedGoogle Scholar
Clark J. M. (1988) Novel nontemplated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucl. Acid Res.
, 9677–9686.CrossRefGoogle Scholar
Landt O., Grunert H.-P., and Hahn U. (1990) A general method for rapid sitedirected mutagenesis using the polymerase chain reaction. Gene
, 125–128.PubMedCrossRefGoogle Scholar
Wang A. M., Doyle M. V., and Mark D. F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA
, 9717–9721.PubMedCrossRefGoogle Scholar
Siebert P. D. and Larrick J. W. (1992) Competitive PCR. Nature
, 557–558.PubMedCrossRefGoogle Scholar
Chomczynski P. and Sacchi E. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.
, 156–159.PubMedCrossRefGoogle Scholar
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
, 2725–2729.PubMedCrossRefGoogle Scholar