METHOD OF ASSESSMENT OF DEGREE OF CELL PROLIFERATION USING MARKER GENES BY METHOD OF POLYMERASE CHAIN REACTION IN REAL TIME Russian patent published in 2013 - IPC C12Q1/68 C12N5/00 

FIELD: biotechnology.

SUBSTANCE: method comprises adding to the part of the wells with lymphocytes of mitogens or test substances, incubation of the contents of the wells, selection of the contents of the wells, cytolysis and isolation of nucleic acids. The reaction of reverse transcription (RT) is carried out with primers for genes tpa and cdc2: primer for RT with mRNA of gene tpa 5'-CATCTTCATCTCACTCTTC-3', primer for RT with mRNA of gene cdc2 5'-CTGGAGTTGAGTAACGAG-3'. Real-time PCR is carried out with the obtained cDNA on the specific primers and with use of the labeled oligonucleotides for genes tpa and cdc2: for gene tpa the forward primer is 5'-TGTCGTGTCAGACCTTGAAGC-3', the reverse primer is 5'-CCTTGGATTTCTTGCTTGTGAC-3', the probe (FAM)-5'-TGTACCACTGTCTCAAGCCCTCCTGC-3'-(BHQ1), for cdc2 gene the forward primer is 5'-CTTCACTTGTTAAGAGTTATTTATAC-3', the reverse primer is 5'-CCAGAGTGTTACTACCTCATGTG-3', the probe (FAM)-5'-TGCCTTGCCAGAGC(FdT)TTTGGAATAC-3'-(BHQ1). The degree of proliferation of lymphocytes is expressed in proliferation index which is the number indicating how many times the amount of mRNA of gene tpa and/or cdc2 increases during culturing lymphocytes with/without addition of PHA or the test substance as compared with the amount of mRNA of the genes in lymphocytes freshly isolated from the blood of healthy donor.

EFFECT: invention enables to monitor directly the number of dividing cells, assessing the degree of expression of the genes involved and regulating the cell division.

4 dwg, 4 ex