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 AR 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 analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp 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 enzymatic 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/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions.
INSERM U439, Pathologie Moléculaire des Récepteurs Nucléaires, Montpellier, France.
P
P
Nirdé
V
V
Georget
B
B
Térouanne
R B
RB
Galifer
C
C
Belon
C
C
Sultan
D013196:Dihydrotestosterone / Q000494:pharmacology; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis*; D011944:Receptors, Androgen / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D012867:Skin / Q000378:metabolism*
D012333:RNA, Messenger; D011944:Receptors, Androgen; D013196:Dihydrotestosterone