QUANTITATION OF ANDROGEN RECEPTOR MESSENGER-RNA FROM GENITAL SKIN FIBROBLASTS BY REVERSE TRANSCRIPTION - COMPETITIVE POLYMERASE-CHAIN-REACTION

To gain further information concerning the regulation by androgen of A R mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain a nalysis of the PCR products for the accurate quantitation of low level s of human androgen receptor (hAR) mRNA in GSF. To control for variati ons due to sample preparation, and to minimize the disparity of the re verse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydro genase (GAPDH) gene, and then adjusted the amount of cDNA to that of t his housekeeping gene. Competitive PCR for hAR was then immediately pe rformed on normalized cDNA with a competitor DNA that exhibited a 13 b p deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis . This quantitation procedure involved no additional steps, such as en zymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/mu g RNA, (+/-1.0, s.e.m.) with an intra- and an inter-a ssay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiologic al concentration of androgen. Quantitative RT-PCR of AR mRNA may be us eful for studying AR mRNA expression in experimental or clinical condi tions. (C) 1998 Elsevier Science Ltd. All rights reserved.