Semi-quantitative reverse-transcriptase polymerase chain reaction: an approach for the measurement of target gene expression in human brain.
Polymerase chain reaction (PCR) is a very powerful tool for qualitative evaluation of nucleic acids due to its high efficiency and convenience. Together with the reverse transcription (RT) reaction, the PCR method has been widely applied to the quantitative measurement of DNA and RNA messages. Since RT-PCR is much more sensitive than all of the traditional methods for quantification of mRNA, including Northern blot, ribonuclease protection, RNA blot, and solution hybridization assays, it is the method of choice for quantitative analyses of low abundance mRNA messages. However, because of the exponential nature of the PCR amplification, RT-PCR quantitation may be problematic, giving false estimates of the abundance of the target messages. By using the constitutively expressed 'housekeeping' gene cyclophilin as a reference gene to normalize mRNA levels, and by taking data from the exponential phase of the PCR amplification, we have developed a rapid and reliable semi-quantitative measurement of the relative abundance of dopamine transporter (DAT) mRNA. The semi-quantitative PCR method has been applied to illustrate its use for the measurement of DAT mRNA in post mortem human brain.
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Department of Neurology, University of Miami School of Medicine, 1501 NW 9th Avenue, Miami, FL 33136, USA.
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D000328:Adult; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D050483:Dopamine Plasma Membrane Transport Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014508:Urea
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C493214:SLC6A3 protein, human; D014508:Urea