Quantifying DNA Methylation in Human HCC

Seventy-seven formalin-fixed, paraffin-embedded human HCC tissue, and 77 corresponding nontumor tissue samples were used for this study. The formalin-fixed, paraffin-embedded tissue specimens were microdissected and analyzed, and DNA was extracted with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), as per the manufacturer's instructions. DNA was modified with sodium-bisulfite using an EZ Methylation-Gold Kit (Zymo Research, CA), and LINE-1 was amplified using a LINE-1-specific polymerase chain reaction. The primer sequences were forward: 5’-TGG TTA AGG GTT TGG GGA TAT T-3’, reverse: 5’-(Biotin)-AAC ACA ATT CCC AAC CCA C-3’, and sequencing: 5’-GGG TTT TGA ATT TGG TA-3’. Quantitative bisulfite pyrosequencing using the PyroMark Q24 Advanced (Qiagen) was performed to quantify the methylation levels of four CpG sites in the repetitive LINE-1 sequence promoter, as described previously.^{[17 (link)]}