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.
نویسندگان
چکیده
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.
منابع مشابه
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 a...
متن کاملQuantitative 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 ...
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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 and deoxycorticosterone (DOC) to their respective 5alpha-reduced derivatives, precursors of allopregnanolon...
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The enzyme 5alpha-reductase (5alpha-R) (EC 1.3.99.5) exists as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2), and both are present in the brain. 5alpha-R1 has been proposed as a constitutive enzyme that essentially plays a catabolic and neuron protective role whereas 5alpha-R2 has been associated with sexually dimorphic functions of the male. In this work, we studied...
متن کاملSteroid 5alpha-reductase isozymes in the adult female rat brain: central role of dihydrotestosterone.
The enzyme 5alpha-reductase (5alpha-R) (EC 1.3.99.5) exists as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2). 5alpha-R1 has been associated with catabolic functions whereas 5alpha-R2 has been associated with sexually dimorphic functions of the male. We recently demonstrated that both 5alpha-R isozymes are present in the central nervous system (CNS) of the adult male ...
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ورودعنوان ژورنال:
- Electrophoresis
دوره 25 3 شماره
صفحات -
تاریخ انتشار 2004