Quantification of steady state expression of mRNA for alpha-1 adrenergic receptor subtypes using reverse transcription and a competitive polymerase chain reaction

MA Scofield, F Liu, PW Abel and WB Jeffries

Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska, USA.

Three distinct alpha-1 adrenergic receptor subtypes (alpha-1a, alpha-1b and alpha-1d) have been identified through molecular cloning. Expression of alpha-1 adrenergic receptor mRNA has been detected in several tissues, but previous studies with Northern blot analysis or RNase protection assays have provided only semiquantitative information. Furthermore, the mRNA distribution of these subtypes in many rat tissues is unknown. In the present study, we quantified alpha- 1a, alpha-1b and alpha-1d adrenergic receptor mRNA in 19 adult rat tissues through the use of reverse transcription (RT) and a competitive polymerase chain reaction (PCR). This procedure used internal cRNA standards as competitive templates that contained sequences complementary to the primers for alpha-1a, alpha-1b and alpha-1d adrenergic receptors and that differed in molecular size from the native sequences. Total RNA was harvested from 19 freshly dissected organs of the rat. The PCR products were labeled by inclusion of alpha- 32P-dCTP in the PCR. After electrophoresis, ratios of competing to native radio-labeled products were used to interpolate the amount of native RNA originally present in each sample. The results revealed that the relative abundance of alpha-1a adrenergic receptor mRNA among various tissues was as follows: vas deferens > colon > or = stomach > or = cerebral cortex > heart > or = small intestine > or = testis > prostate. The relative rank order of alpha-1b adrenergic receptor mRNA expression was heart > or = cerebral cortex > liver, whereas that of alpha-1d adrenergic receptor mRNA was vas deferens > cerebral cortex > or = aorta > adrenal gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Volume 275, Issue 2, pp. 1035-1042, 11/01/1995
Copyright © 1995 by American Society for Pharmacology and Experimental Therapeutics

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