Quantitation of mRNA levels of steroid 5alpha-reductase isozymes in the rat brain by "one-step" RT-PCR and capillary electrophoresis

Abstract

The enzyme 5alpha-reductase (5alpha-R) is present in many mammalian tissues, including the brain. The physiological importance of 5alpha-R in the brain derives from its capability to convert testosterone (T) to a more potent androgen, dihydrotestosterone (DHT), and to convert progesterone to its 5alpha-reduced derivative, precursors of allopregnanolone, potent allosteric modulator of the gamma-aminobutyric acid receptor (GABA(A)-R). 5alpha-R occurs as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2). We present an accurate, rapid, and modestly labor-intensive method to precisely quantitate 5alpha-R mRNA species in the cerebral cortex of the rat. This approach combines the high specificity of "one-step" reverse transcription-polymerase chain reaction (RT-PCR) with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). Both cDNA synthesis and PCR amplification are performed with the same enzyme and site-specific primers, improving the efficiency of cDNA synthesis. The specific target mRNA and a mimic DNA fragment, used as a competitive internal standard, were co-amplified in a single reaction in which the same primers are used. The method presented in this paper enables a more efficient quantitative determination of 5alpha-R mRNA isozymes, and may lead to a better understanding of the role of 5alpha-R isozymes in the physiology of the central nervous system.