EA033170B1 - Method for gstm1 gene expression detection in samples of paraffin-fixed sections of mammary tumor tissue - Google Patents

Abstract

The invention relates to medicine, in particular, to clinical laboratory diagnostics, and may be used for detecting the GSTM1 gene in samples of paraffin-fixed sections of mammary tumor tissue. The invention objective is to develop a method for detecting the GSTM1 gene expression in samples of paraffin-fixed sections of mammary tumor tissue, to provide the possibility of assessing the efficiency of the 2nd phase of xenobiotic biotransformation in the tumor cells (assessing the cells' ability of biochemically inactivating reactive xenobiotic metabolites). The objective is achieved as follows: human RNA is released from the paraffin-fixed sections of mammary tumor tissue, a reverse transcription reaction is conducted to obtain cDNA, which is then used for performing real-time TaqMan PCR with the use of matched pairs of primers and probes: for GSTM1 gene: forward-primer AAGTTGGCATGCCCATGATACTGG, reverse-primer CGTCCCCCATCGTGTACTTCTTTTC, TaqMan probe FAM-CCATCCGCCTGCTCCTGGAATA-BHQ1; for the reference gene HGUS: forward-primer CTCCAAGGAATTTTGCCGATT, reverse-primer GGCGTGAAGATCCGGTTTTTA, TaqMan probe JOE TGTTCAGTCACCGACAGTGCTG-BHQ2, the primers and the probes being introduced in a concentration of 3.2 pmol/μl, and then amplification is conducted according to the following programme: 1 cycle: 95°C - 5 min.; 50 cycles: 95°C - 20 s, 62°C - 20 s, 72°C - 15 s; 1 cycle: 72°C - 10 min, fluorescence detection is conducted at FAM and JOE channels; if the tested material has threshold cycle (Ct) values in the range of 0-20 cycles or 36-50 cycles for the JOE channel (reference gene HGUS), it is concluded that human cells are absent in the tested material and that GSTM1 gene expression cannot be assessed in the sample; if the tested material has Ct values in the range of 21-35 cycles for the JOE channel, it is concluded that human cells are present in the sample and then fluorescence data analysis is conducted for the channel FAM (gene GSTM1), if Ct values in the range of 21-35 cycles are determined for the FAM channel, a conclusion on the presence of GSTM1 gene expression is made, and if the tested material has Ct values in the range of 0-20 cycles or 36-50 cycles for the FAM channel, a conclusion is made on the absence of GSTM1 gene expression.