Abstract:

Robust standardized methods to extract DNA from formalin-fixed paraffin-embedded tissues are required for accurate bisulfite conversion based methylation analysis.Despite many different protocols are published, extraction of sufficient quantity of intact DNA from formalin-fixed paraffin-embedded (FFPE) tissues is still challenging. We compared two DNA extraction methods (Phenol-Chloroform and Qiagen mini kit) in terms of DNA yield, concentration, purity and devised a modified protocol of Qiagen minikit. DNA fragment size and amplification rate were assessed with GAPDH 499 bp and BRCA1 non-methylated 86 bp primers. Efficiency of bisulfite conversion and subsequent methylation-specific PCR were assessed with BRCA1 methylated 75 bp primers.The modified protocol of Qiagen minikit yielded a sufficient quantity of purified DNA for methylation analysis than phenol chloroform protocol. Each modified parameter excluding age of tumor blocks had significant (P<0.05) effect on extracted DNA. Amplification rate was high with short amplicons (86 bp) than large amplicons (499 bp).100% amplification rate of touchdown methylation-specific PCR (MSPCR) was found with BRCA1 75 bp primers.The devised modified protocol of Qiagen minikit coupled with touchdown MSPCR are suitable and cost-effective methods for wide-range of downstream molecular and epigenetic applications.

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