Quantitative reverse-transcriptase polymerase chain reaction assay for mRNA levels of steroid 5alpha-reductase isozymes.

Many genes that play a major physiologic role present low levels of expression, whose characterization requires accurate quantitation methods of adequate sensitivity. The use of the reverse-transcriptase polymerase chain reaction (RT-PCR) coupled to capillary electrophoresis (CE) for the analysis of steady-state messenger RNA levels has become increasingly widespread in recent years [1–5]. The present paper describes a rapid and accurate RTPCR method coupled to CE to quantitate the mRNA expression of the two 5a-reductase (5a-R) (EC 1.3.99.5) isozymes, 5a-reductase type I (5a-RI), and 5a-reductase type II (5a-RII), which play an important role in virilization in mammals [6]. This method combines the high degree of specificity of competitive PCR with the sensitivity of capillary electrophoresis. Total RNA isolated from the tissue was reverse transcribed (problem cDNA) and mixed with a known amount (number of molecules) of a synthetic internal standard (mimic DNA). Both standard and unknown problem cDNA were then subjected to PCR amplification using the same sets of primers. The amount of the mRNA in question was quantitated by comparing the fluorescence intensity of the problem cDNA with that of a known amount of the internal standard after separation by capillary electrophoresis.