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 use d to detect low abundance mRNAs in small samples. Accurate quantitativ e measurement of their level, as required for the study of gene expres sion, can be performed by RT-competitive PCR, a method that relies on the addition of known amounts of a cDNA competitor molecule in the amp lification reactions. Here we demonstrate that this method can be easi ly set up in any laboratory with a minimum of equipment in molecular b iology, and that either homologous or heterologous competitor, with a small difference in sequence length relative to the target, can be use d to quantify specific mRNA accurately. We propose the utilization of a thermostable reverse transcriptase in the RT step to overcome the pr oblem of the efficiency of target cDNA synthesis. In addition, to obta in reliable measurements, we recommend performing four PCR reactions w ith amounts of competitor flanking the concentration of the target mRN A. (C) 1997 Academic Press.