Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in reverse...

We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.