METHOD OF DETERMINING LEVEL OF GENE TRANSCRIPTION, WHICH CODES HUMAN CHEMOKINES CCL2 (-1) AND SET FOR ITS REALIZATION

摘要

1. A method for determining the level of transcription of the gene encoding the chemokine CCL2 (MCP-1) Human comprising: isolation of RNA from the test material samples (blood, tissue, koskoby, punctate, cell cultures) cDNA synthesis by reverse transcription reactions, amplification of gene cDNA with the aid of real time PCR, characterized in that: a) the normalization of the results is performed by a standardizing factor, calculated as the average number of genes in the cDNA sample RPL32, meetwithlove.com This user and PII encoding ribosomal protein L3 2, b-actin and polymerase-II respectively, the amplification RPL32 gene cDNA meetwithlove.com This user and PII performed using a triplex-PCR, and b) the accumulation of amplification products are detected in real time using one of the following: 1b) using intekaliruyuschego double-stranded DNA fluorescent dye with a high index of discrimination in fluorescence bound and unbound forms, e.g., SYBR GreenI (Invitrogen); 2b) using an oligonucleotide probe complementary type TaqMan central portion of the amplified fragment of the transcript; sample length of 20-30 nucleotides, carries at the 5 'end of the fluorophore (FAM, R6G, ROX) and at the 3' end is not a fluorescent quencher (BHQ, FTQ et al.); c) determining the relative amount of cDNA gene MSR1 and normalizing genes RPL32, meetwithlove.com This user and PII is carried out using a calibration curve constructed with standard samples of DNA amplification taken in 6 sequentially decreasing threefold dilutions of known concentrations; represents a standard mixture of DNA fragments carrying the sequence of the amplified gene RPL32 cDNA meetwithlove.com This user, PII MSR1 and a length of 500 bp; about