Quantitation of mRNA levels of steroid 5alpha-reductase isozymes: a novel method that combines quantitative RT-PCR and capillary electrophoresis.
A novel, accurate, rapid and modestly labor-intensive method has been developed to quantitate specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR). This strategy combines the high degree of specificity of competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). The specific target mRNA and a mimic DNA fragment, used as an internal standard (IS), were co-amplified in a single reaction in which the same primers are used. The amount of mRNA was then quantitated by extrapolation from the standard curve generated with the internal standard. PCR primers were designed to amplify both a 185 bp fragment of the target cDNA for steroid 5alpha-reductase 1 (5alpha-R1) and a 192 bp fragment of the target cDNA for steroid 5alpha-reductase type 2 (5alpha-R2). The 5' forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). Two synthetic internal standard DNAs of 300 bp were synthesized from the sequence of plasmid pEGFP-C1. The ratio of fluorescence intensity between amplified products of the target cDNA (185 or 192 bp fragments) and the competitive DNA (300 bp fragment) was determined quantitatively after separation by capillary electrophoresis and fluorescence analysis. The accurate quantitation of low-abundance mRNAs by the present method allows low-level gene expression to be characterized.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Granada, Avenida de Madrid s/n, 18012, Granada, Spain.
Jesús M
JM
Torres
Esperanza
E
Ortega
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; D012680:Sensitivity and Specificity
D007527:Isoenzymes; D012333:RNA, Messenger; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase