Improved results of LINE-1 methylation analysis in formalin-fixed paraffin-embedded tissues with the application of a heating step during the DNA extraction process

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes. However, formalin-fixation introduced inter-strand cross-linking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. To identify whether formalin fixation impact the measured values of methylation in LINE-1 repetitive elements and whether heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, LINE-1 methylation levels of paired fresh-frozen (FF) and FFPE mouse xenograft tissue samples was measured by pyrosequencing assay. Moreover, to further confirm the effect of additional heating step during the DNA extraction in the measurement of LINE-1 or single gene methylation levels, FFPE gastric cancer and colorectal cancer patient samples were analyzed for the methylation status of LINE-1 and single genes. Intriguingly, formalin fixation per se led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Application of heating of the DNA at 95°C for 30 min before bisulfite conversion was found 1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, 2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and 3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer.<br/> <br/> In conclusion, application of heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples. <br/>