Development of a Qualitative Detection Method for RASSF1A Gene Methylation Based on Fluorescence PCR
Objective:This paper employs high-throughput sequencing to assist in the development of a qualitative detection kit for the methylation of ras-association domain family 1A(RASSF1A)gene(fluorescence PCR method).Methods:DNA from 37 lung cancer plasma samples is extracted and subjected to bisulfite modification.High-throughput sequencing is used to detect the efficiency of bisulfite conversion and the methylation levels at CpG sites of the RASSF1A gene.The methylation status of the RASSF1A gene in the samples is also detected using the fluorescence PCR method.Results:The high-throughput sequencing shows that average conversion efficiency of sample DNA is 99.47%;based on the results of the two-way hierarchical clustering analysis of the methylation levels at CpG sites,CpG sites 26 to 30 are selected as the detection sites for fluorescence PCR method.With a methylation level threshold of 5%,and the ΔCt Cut-off value of 9,the results of the two detection methods are consistent for the 37 samples.Conclusion:This study successfully establishs a qualitative detection method for RASSF1A gene methylation based on the fluorescence PCR method with the aid of high-throughput sequencing technology.The method is easy to operate,cost-effective,and efficient,making it suitable for routine clinical detection of the methylation status of the RASSF1A gene.
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万方数据
维普
