FFPE DNA Methylation Analysis

CpG methylation analysis was performed using Infinium 850k Methylation array (Illumina) according to the manufacturer’s recommendations. Briefly, 200 ng of DNA extracted from each sample was utilized to restore the degraded FFPE DNA to a state that is analyzable by the Infinium HD FFPE whole genome amplification workflow as described in the Infinium FFPE Restoration guide (Illumina). DNA was denatured using 8 µL of NaOH 0.1N for 10 min at room temperature. A 1 h reaction at 37°C was then performed with Primer Pre-Restore (PPR) and Amplification Mix Restore (AMR) reagents supplied by the kit manufacturer in which DNA repair is accomplished. Restored DNA was cleaned using a ZR-96 DNA Clean and Concentrator-5 kit (Zymo Research) following the manufacturer’s protocol. DNA was concentrated between 150 ng/uL of concentration. Between 100 and 200 ng of prepared DNA was used as input for the hybridization on the Illumina 850K Epic BeadChip array and processed according to kit instructions. Data were quality assessed using Illumina Genome Studio software (Illumina), and idat files were uploaded to the DKFZ Heidelberg Classifier v12.5 (www.molecularneuropathology.org).^{16 (link)}