Optimizing The Bisulfite Conversion Method And Its Application In The Clinical Detection Of Gastric Cancer Related RPRM Gene Methylation

The bisulfite conversion technique is a widely accepted method used for DNA 5-methylcytosine detection.With bisulfite treatment,unmethylated cytosines in DNA are deaminated to uracil,while 5-methylcytosines are resistant to this transformation.However,the conventional procedures for bisulfite conversion is time-consuming and has a high DNA degradation rate.Therefore,our purpose is to improve the conventional procedures to achieve a high efficiency of cytosine conversion with lower DNA degradation and shortened reaction time.Then we applied this modified method to the analyses of RPRM gene methylation in gastric cancer.Based on methylation-sensitive melt curve analyses,we assessed the impacts of different bisulfite salts,high temperature denaturation conditions,conversion reaction temperature,antioxidants and denaturants on DNA conversion and degradation.The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing.Then we applied the modified method to the analysis of the methylation status of RPRM gene promoter in genomic DNA from gastric cancer samples including cell lines,tumor tissues,paracancerous tissue,plasma and peripheral blood mononuclear cell(PBMC),to determine the correlation between RPRM gene methylation and gastric cancer.According to our study,we demonstrate that high temperature denaturation is a primary reason for DNA degradation,and the addition of ethylene glycol dimethyl ether(DME)is an effective way to accelerate the bisulfite conversion process.In summary,the modified method uses 4 M NaHSO3 and 10%DME to convert DNA under the condition of 95? 3 min,(95? 30 s,70? 10 min)X 12 cycles.The conversion process can be completed within 2 hours.The conversion efficiency is comparable to many other commercial kits,thus it has a great potential for the DNA methylation research.In the study of RPRM gene methylation in gastric cancer,a high degree of RPRM gene promoter methylation in cell lines and gastric cancer tissues was found,indicating a strong correlation between RPRM methylation and gastric cancer.A certain degree of methylation was also detected in the gastric cancer plasma samples,suggesting that the detection on methylation of RPRM gene promoter in human plasma may be helpful for gastric cancer diagnosis.