METHOD FOR DETERMINING LEVEL OF EXPRESSION OF GENE ENCODING CCL-2 IN TISSUES OF EYE OF RABBIT ORYCTOLAGUS CUNICULUS, AND KIT FOR ITS DETERMINATION Russian patent published in 2021 - IPC C12N15/00 C12Q1/68 

FIELD: medical biotechnology.

SUBSTANCE: invention relates to the field of medical biotechnology. The invention provides a method for determining the level of expression of a gene encoding CCL-2 in tissues of a rabbit eye (Oryctolagus cuniculus) using real-time RT-PCR and a kit for its determination. When implementing the method, the following is carried out: a) the use of one reference GAPDH gene to normalize the results obtained; b) selection of original primer pairs for the investigated and reference genes: CCL2_1 F-5'-ATGAAGGTCTCTGCAACGCT-3', CCL2_1R-5'-CCCTTGGCCAGTTTGGTCАТ-3', GAPDHF-5'-GATTGTCAGCAACGCATCCTG-3', GAPDH_R-5'-CTCCACAATGCCGAAGTGGT-3'; c) homogenization of eye tissues for 90 s at a speed of 45,000 rpm; d) isolation of mRNA from samples with subsequent addition of 1 mcl of RNase inhibitor to the sample; e) synthesis of cDNA by reverse transcription; f) amplification of the studied and reference genes using real-time PCR with fixation of fluorescence using the intercalating dye SYBR Green (Evrogen); g) determination of the relative amount of the CCL-2 gene and the reference GAPDH gene using a calibration curve constructed during amplification of a sample with a known DNA concentration, five times diluted 4 times; the amplification efficiency, determined by the equation of the calibration curve, is used in calculating the relative magnitude and normalized expression of the gene of interest. The kit for determining the level of expression of the gene encoding the CCL-2 of the rabbit Oryctolagus cuniculus includes: a) original oligonucleotide primers for the CCL-2 gene of the rabbit Oryctolagus cuniculus: CCL21F-5'-ATGAAGGTCTCTGCAACGCT-3', CCL2_1R-5'-CCCTTGGCCAGTTTGGTCAT-3'; b) original oligonucleotide primers for the GAPDH gene of the rabbit Oryctolagus cuniculus: GAPDHF-5'-GATTGTCAGCAACGCATCCTG-3', GAPDH_R-5'-CTCCACAATGCCGAAGTGGT-3'; c) the reaction mixture of PNR in real time for amplification of the CCL-2 gene, containing: distilled water 9.4 mcl, buffer 1.5 mcl (Evrogen), forward primer CCL-2_F (concentration 1 mcM; Mw 60.98 g / mol) 1 mcl, reverse primer CCL-2_R (concentration 1 mcM; Mw 60.56 g / mol) 1 mcl, dNTP 0.5 mcl, cDNA 1 mcl, SYBR Green I [1: 25000] 0.3 mcl, Taq -polymerase (0.5 unit / mcl) 0.3 mcl (Biosan); d) a real-time PCR reaction mixture for amplification of the GAPDH gene, containing: distilled water 9.4 mcl, buffer 1.5 mcl (Evrogen), direct primer GAPDH_F (concentration 1 mcM; Mw 63.86 g / mol) 1 mcl , reverse primer GAPDH_R (concentration 1 mcM; Mw 60.83 g / mol) 1 mcl, dNTP 0.5 mcl, cDNA 1 mcl, SYBR Green I [1: 25000] 0.3 mcl, Taq polymerase (0.5 units / mcl) 0.3 mcl (Biosan); e) protocol for the amplification of the CCL-2 and GAPDH genes, including: primary denaturation - 3 min, 95°C, amplification cycle (x45), denaturation - 15 s, 95°C, primer annealing - 20 s, 56.2°C , elongation and removal of the fluorescent signal - 30 s, 72°C.

EFFECT: invention ensures obtaining a protocol for sample preparation, setting and calculating the results of RT-PCR with an optimally selected set of reagents, allowing high accuracy to reproduce the reaction under the conditions of PCR laboratories, for further use in experimental ophthalmology, in preclinical studies of medical devices and, in particular, when testing anti-inflammatory drugs.

2 cl, 3 dwg, 1 tbl, 1 ex