Quantitative reverse transcriptase polymerase chain reaction assay for mouse androgen receptor mRNA

A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) ass ay for mouse androgen receptor (AR) mRNA was developed to study relative ch anges in AR gene expression. Serial dilutions of a standard comprising a fr agment of the ampicillin resistance gene flanked by the primer sequences of the AR mRNA were added to a constant amount of total RNA for RT-PCR. Prime rs were designed to generate a 541-bp fragment of mouse AR mRNA (target [T] ) and a 460-bp fragment of the standard (S). PCR products were resolved by gel electrophoresis and quantitated by densitometry. A standard curve was g enerated for each sample by plotting the logarithm of T/S products vs the l ogarithm of the amount of S added. The amount of T was determined from the standard curve where intensities of PCR products of T and S were equal. The assay was validated by measuring the relative abundance of AR mRNA in 10 m ouse tissues, and results were consistent with studies of AR expression in rat tissues. Assay reproducibility, tested by repeating assays on four diff erent tissues on different days from the RT step, had a coefficient of vari ation of 6-16%. The current assay is thus both reproducible and valid in qu antitation of mouse AR mRNA.