Methylation specific PCR (MSP)

Purified genomic DNA (200 ng) was subjected to bisulfite conversion using the EZ DNA Methylation Kit (Zymo Research), following manufactures instructions. MSP was carried out using KAPA LongRange HotStart DNA Polymerase (Sigma-Aldrich) and included 1X KAPA LongRange Buffer, 6.7 mM of MgCl₂, 1.25 mM KAPA dNTP mix, 300 ng of each primer, 1.25U of KAPA LongRange HotStart DNA Polymerase, 50 ng genomic DNA, in ultra-pure water. For each DNA sample, two distinct PCR reactions were performed using the MGMT primer pairs outlined in Supplementary Table ³, as previously described²⁰. Briefly, the MGMT promoter contains 98 CpG sites, which have been assigned consecutive numbers (5′ to 3′) starting with CpG1 (− 452 bp from transcriptional start site) through to CpG98 (+ 308 bp). CpGs 73–90 play a critical role in the transcriptional control of MGMT expression^64,65, and were thus targeted for our MSP approach (9 CpGs in total). Each primer pair was specific to either methylated or unmethylated DNA sequences. Positive control DNA was the EpiTect PCR Control DNA set (Qiagen, Hilden, Germany), which included completely unmethylated human genomic DNA, completely unmethylated bisulfite converted human genomic DNA, and completely methylated bisulfite converted human genomic DNA. Unmethylated human genomic DNA was subjected to bisulfite conversion in parallel to the genomic DNA extracted from patient tissue. Negative controls without DNA were performed for each of the methylated and unmethylated primer reactions. Thermal cycling conditions, performed on the Mastercycler pro PCR system (Eppendorf, Hamburg, Germany), included an initial denaturation step at 95 °C for 5 min, 35 cycles of denaturation (95 °C, 30 s), annealing (59 °C, 30 s) and extension (72 °C, 30 s), and a final extension at 72 °C for 4 min. PCR products were visualised on 2% agarose gels (agarose, TAE buffer and SYBR Safe DNA Gel stain (Life Technologies, Carlsbad, CA, USA)).