Quantitation of mRNA levels of steroid 5alpha-reductase isozymes: A method that combines one-step reverse transcription-polymerase chain reaction and separation by capillary electrophoresis.
We developed an accurate, rapid, and modestly labor-intensive method to precisely quantitate mRNA species by one-step reverse transcription-polymerase chain reaction (RT-PCR). This approach combines the high specificity of quantitative competitive 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 coamplified in a single reaction in which the same primers are used. The 5' forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). The ratio of fluorescence intensity between amplified products of the target cDNA and the competitive DNA was determined quantitatively after separation by CE and fluorescence analysis. Using this method, we have been able to precisely quantify the mean amount of steroid 5alpha-reductase (5alpha-R) isozyme mRNA levels in ventral prostate of the rat, detecting 10-fold difference for 5alpha-R1 and 50-fold difference for 5alpha-R2, respectively, in comparison with our previously reported two-step method. Because the competitive RT-PCR presented in this paper enables a more efficient quantitative determination of mRNAs, low-level gene expression could be quantified.
Torres
Jesús M
JM
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Granada, Spain.
Ortega
Esperanza
E
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000032:analysis*; D000818:Animals; D019075:Electrophoresis, Capillary; D007527:Isoenzymes / Q000032:analysis; D008297:Male; D011467:Prostate / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D012680:Sensitivity and Specificity
D007527:Isoenzymes; D012333:RNA, Messenger; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase