Tissue Extraction and DNA Isolation from FFPE Samples

Independent control of histopathology, tumor cell content of routine pathological specimens, and the selection of tissue areas for tissue extraction were determined by the pathologist. Subsequently, cylinders 1.5 mm in length and 2 mm in height were stamped out from the formalin-fixed and paraffin-embedded (FFPE) tissue blocks using an 18 Charrière core stamp and subjected to DNA isolation. Genomic DNA was extracted using the automated MagNA Pure LC 2.0 system and MagNA Pure LC DNA isolation kit II - tissue (Roche Diagnostics Deutschland, Roche Applied Science, Mannheim, Germany). The quality of extracted DNA was assessed using spectrophotometry, gel electrophoresis, and quantitative PCR, which characterized the yield, purity, and grade of degradation of isolated DNA. Bisulfite conversion of DNA was carried out using the EZ DNA Methylation-Gold Kit (Zymo Research Corporation, Irvine CA, USA) and 1 µg of isolated DNA. Fully methylated and converted DNA, as well as unmethylated bisulfite-converted DNA controls, were used as reported previously [21] (link).