Investigation of GSTP1 promotor region methylation in peripheral bloods of prostate cancer

In this project, it was investigated whether it is possible to analyse the promoter region methylation of GSTP1 gene in peripheral blood. For this purpose, DNA was isolated from peripheral blood of prostate cancer patients and the promoter region methylation of GSTP1 gene was examined by using the obtained isolate. After bisulfite modification of the isolated DNAs, Methylation Sensitive High-Resolution Melting (MS-HRM) was performed using primer pairs designed for methylated and unmethylated DNA sequences. As a result, when MS-HRM method applied to the peripheral blood DNA isolates, GSTP1 promoter methylation could not detected due to due to the presence of excessive amount of unmethylated leukocyte gDNA. The same DNA isolates were examined by ddPCR, which is known to be one of the most accurate and reliable tools due to its high sensitivity and specificity and 2 of 65 samples were detected as GSTP1 methylated. In this study, the third method used to detect GSTP1 promoter methylation was Nested MS-PCR method which consists of Nested PCR and MS-HRM. The purpose of this method is to increase the number of copies of methylated GSTP1 fragments released from the tumour cells which has very low concentration compared to unmethylated GSTP1 fragments of leukocyte gDNA and to increase the sensitivity and specificity of the MS-HRM to be performed to detect methylation. With this method, methylation was detected in the promoter region of GSTP1 gene in 9 of 65 samples.As a result of this study, although the sample could not be collected in accordance with ctDNA studies, it was shown that it is possible to detect promoter region methylation using appropriate methods.Bu yöntem ile toplamda 65 örnekten 9 tanesinde GSTP1 geni promotor bölgesinde metilasyon varlığı saptanmıştır. Bu çalışmanın sonucunda ctDNA çalışmalarına uygun olarak örnek toplanamamış olsa da uygun yöntemler kullanılarak promotor bölge metilasyonunun saptanabileceği gösterilmiştir.