FIELD: biotechnology and medicine.
SUBSTANCE: diagnostics is performed using oligodeoxyribonucleotide primers and a fluorescently labeled probe corresponding to one of the primer pairs, complementary to the regions of the detected fragments, as well as using standard samples, which are a series of cDNA dilutions of the characterized calibrator sample. RNA is extracted from whole blood or dried blood spots (dried drops of blood), reverse transcription is performed to obtain cDNA, after which the resulting cDNA sample is used for simultaneous amplification in one container of sections of the target gene IFNAR-1 and two reference normalization genes HPRT and RPP30, and also for amplification of IFNAR-1 gene transcript fragments in other containers, followed by Sanger sequencing. When determining the level of expression for amplification, the results obtained are recorded using the software of the device used to carry out PNR with hybridization-fluorescence detection in real time. Analysis of IFNAR-1 expression is performed with normalization to reference genes, and a “normalization coefficient” is introduced, calculated as the geometric mean or arithmetic mean of the values of all reference normalization genes. The relative expression level of IFNAR-1 was determined using the methodΔΔCT in comparison with a series of cDNA dilutions of a characterized calibrator sample, which contains the wild-type IFNAR-1 gene, based on which conclusions are drawn about the normal or reduced level of expression of the target gene. When determining the nucleotide sequence of the complete IFNAR-1 gene transcript, two overlapping transcript fragments are amplified: 5,944 nucleotides long, region 1–5944 nt., and 4,607 nucleotides long, region 1,551–6,157 nt., according to the reference sequence presented in the international GenBank database (NM_0013 84504.1). Then direct Sanger sequencing of the resulting fragments of the IFNAR-1 gene transcript is carried out and conclusions are drawn about the presence or absence of potentially pathogenetically significant mutations. If reduced expression of IFNAR-1 and/or mutations in the nucleotide sequence of the transcript are detected, leading to changes in the amino acid sequence of the gene product, it is concluded that the subject has a predisposition to the development of HCC.
EFFECT: ensuring an increase in the sensitivity and reliability of diagnosis, a decrease in false-positive (low levels of IFNAR-1 gene expression) results, and therefore an expansion of the capabilities of the method, an expansion of the arsenal of tools used to predict the development of hepatocellular carcinoma in viral hepatitis.
4 cl, 3 dwg, 3 tbl, 3 ex
