METHOD AND MEANS FOR QUANTITATIVE ANALYSIS OF THE NUMBER OF MOLECULES CODING FOR DIFFERENT GENES IN CELLS

摘要

The invention concerns a method for in vitro and/or ex vivo quantitative analysis of the number of nucleic acid molecules coding for different genes expressed in cells of living organisms, on a small number of cells, preferably on a single cell, which consists in: performing a reverse transcription and a first polymerase chain reaction (RT-PCR) of mRNA of a small number of lyzed cells, separating the various products of the first polymerase chain reaction to individualize the genes, and performing a second polymerase chain reaction on the resulting separated individual genes. While the first polymerase chain reaction is carried out by means of an association of 5' primers and 3' primers, said 3' primers being identical to the 3' primers used for the reverse transcription, the method consists in using, as reference for calculating the number of copies of genes generated by polymerase chain reaction, the individual cellular DNA coding for the gene concerned, controlling the efficacy of the reverse transcription, by means of a polymerase chain reaction of the cDNA's of one of the genes concerned, in parallel to the same amplification of the mRNA molecules of the same gene, on cellular samples containing the same number of molecules, and evaluating the results of said second polymerase chain reaction.