The use of the reverse transcription-competitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples.
Reverse transcription-polymerase chain reaction (RT-PCR) is widely used to detect low abundance mRNAs in small samples. Accurate quantitative measurement of their level, as required for the study of gene expression, can be performed by RT-competitive PCR, a method that relies on the addition of known amounts of a cDNA competitor molecule in the amplification reactions. Here we demonstrate that this method can be easily set up in any laboratory with a minimum of equipment in molecular biology, and that either homologous or heterologous competitor, with a small difference in sequence length relative to the target, can be used to quantify specific mRNA accurately. We propose the utilization of a thermostable reverse transcriptase in the RT step to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, we recommend performing four PCR reactions with amounts of competitor flanking the concentration of the target mRNA.
Auboeuf
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INSERM U449, Faculté de Médecine R. Laënnec, Lyon, France.
Vidal
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D000818:Animals; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005069:Evaluation Studies as Topic; D005786:Gene Expression Regulation; D051275:Glucose Transporter Type 4; D006801:Humans; D020738:Leptin; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D009124:Muscle Proteins; D016133:Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics; D015203:Reproducibility of Results; D014158:Transcription, Genetic
D051275:Glucose Transporter Type 4; D020738:Leptin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011506:Proteins; D012333:RNA, Messenger; C495692:SLC2A4 protein, human; C495694:Slc2a4 protein, rat; D012313:RNA; D011972:Receptor, Insulin; D012194:RNA-Directed DNA Polymerase