Prevalence of mutations in codons 12 and 13 K- RAS2 gene by Cold Real Time PCR method in colorectal cancer patients referred to Imam Khomeini Hospital

Background and Purpose: Colorectal cancer is the most prevalent cancers in Western societies. Given the prevalence of these tumors in the country and access to treatment based on tumor biology, identifying genetic changes is necessary. The aim of this study was to evaluate rapid and sensitive method for screening gene mutations using COLD Real-Time PCR KRAS in samples of patients. Material and Method: Genomic DNA samples from 17 patients with colorectal cancer tissue in paraffin blocks were extracted. Determination of the K-RAS gene mutation for exon 2, was performed by Real-Time PCR using hybridization probes designed to codons 12 and 13, respectively. DNA extraction accuracy by identifying Beta- actin gene was confirmed in all cases. Sensitivity COLD Real-time PCR with serial dilutions of cell lines SW-480 and HCT-116 was determined. Findings: In this study of 17 paraffin blocks were evaluated that in 15 blocks Beta actin gene was identified with the 500 bp. five cases of patients with the K-RAS gene were heterozygous form and in 7 samples of patients with changes in DNA concentration Beta- actin- gene was identified d. sensitivity of COLD Real-time PCR was determined with serial dilution of approximately 280 cells per microliter for cell line SW-480 (codon 12) and 27 cells cells per microliter for in cell line HCT-116 (codon 13).Discussion and conclusion: Failure to identify reference genes (Beta actin) as an indicator of problems with the quality extraction of DNA from paraffin blocks and thus in detection of K-RAS mutations. When the length of PCR product is over 500 bp, in addition to the existence of inhibitors, the degradation of nucleic acid extraction during was shown. Changes the in DNA concentration is appropriate, in cases where the pattern of PCR Beta acting gene is as smear. The results of serial dilutions of cell lines indicate proper sensitivity of COLD Real-Time PCR K-RAS codon mutants. The results of this method of counting the time needed to extract less than an hour. Search in order to use this method and fixative of non-formalin in biopsy specimens during the study is processing.

کلیدواژه ها:

Codon 12 & 13 ، Colorectal cancer ، KRAS