Quantitation of mRNA levels of steroid 5 alpha-reductase isoenzymes in the rat brain by "one-step" RT-PCR and capillary electrophoresis.
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.
Torres
Jesús M
JM
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Granada, Spain.
Sánchez
Pilar
P
Ortega
Esperanza
E
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000032:analysis*; D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis; D008297:Male; D017397:Prefrontal Cortex / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D007527:Isoenzymes; D012333:RNA, Messenger; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase