Quantitation of mRNA Species by RT-PCR on Total mRNA Population with Nonradioactive Probes

Quantitative polymerase chain reaction (PCR) is aimed to determine the absolute or relative amounts of RNA or DNA sequences in a given sample. There are two facts limiting the convenience of this approach. First, in most cases, only one or two sequences are amplified in a given round of amplification. If a family of sequences are to be quantitated, as many amplification reactions are necessary. However, it has been shown that complex populations could be amplified in a sequential independent way (1 –3 ). A major concern about the amplification of whole populations are the biases for or against some sequences. In fact, it appears that these biases are not important and that the amplified populations are quite representative of the original mixture of sequences (1 ,4 ). This makes possible a score of PCR applications such as differential display analysis (5 ) or representational difference analysis (6 ), which are aimed to detect qualitative and quantitative differences between sequences present in genomes or messenger RNA (mRNA) populations. This also implies that it is possible to measure the amount of numerous sequences in the amplicons.