METHOD FOR DETERMINING THE LEVEL OF EXPRESSION OF THE GENE ENCODING IL-18 IN THE TISSUES OF THE EYE OF THE ORYCTOLAGUS CUNICULUS RABBIT, AND DETERMINATION KIT Russian patent published in 2021 - IPC C12N15/00 C12Q1/68 

FIELD: biotech.

SUBSTANCE: invention relates to the field of medical biotechnology and relates to a method for determining the level of expression of a gene encoding IL-18 in tissues of a rabbit eye (Oryctolagus cuniculus), and a kit for its determination. The method includes: a) the use of one reference GAPDH gene to normalize the results obtained, b) selection of original primer pairs for the studied and reference genes, c) homogenization of eye tissues for 90 seconds at a speed of 45,000 rpm, d) isolation of mRNA from samples with the subsequent addition of 1 μl of an 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 fluorescence fixation using the intercalating dye SYBR Green (Eurogene), g) determination of the relative amount of the gene IL18 and the reference GAPDH gene using a calibration curve constructed from amplification of a sample with a known DNA concentration, five-fold diluted 4-fold. 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 includes: a) original oligonucleotide primers for the IL1β gene of the rabbit Oryctolagus cuniculus

IL1β_F-5'-ACCAGAAGAGGCTTGCATCA - 3'

EFFECT: invention makes it possible to obtain a protocol for sample preparation, formulation and calculation of RT-PCR results with an optimally selected set of reagents, which allow to reproduce the reaction with high accuracy 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, 2 dwg, 1 tbl, 1 ex