Cpgenome universal methylated dna and unmethylated dna

To validate the aberrantly methylated loci, we performed bisulfite pyrosequencing. About 150 ng DNA in the discovery and validation sets was bisulfite converted using EZ DNA Methylation-Lightning™ Kit (Zymo Research). The completeness of bisulfite conversion was evaluated using Calponin PCR 26 (link). The bisulfite-converted DNA was amplified using biotin-labeled PCR primers. The custom-designed PyroMark assay IDs (Qiagen) and PCR conditions for the selected loci are listed in Table S3. Bisulfite-converted CpGenome universal methylated DNA and unmethylated DNA (Millipore, Billerica, MA) were used as methylated and unmethylated controls. Bisulfite pyrosequencing was performed by the Centre of Genomics Sciences in the University of Hong Kong using PSQ96MA (Biotage, Charlotte, NC) as previously described 27 (link).

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was used to evaluate MGMT promoter methylation status in paraffin embedded tumor samples. DNA was extracted from paraffin sections of glioblastoma patients using the Qia-Amp DNA mini kit (Qiagen) after deparaffinization. MS-MLPA was performed using the MS-MLPA probe mix prepared by Salsa MS-MLPA Kit ME011 MMR (MRC-Holland), as described by the manufacturer. After denaturation of the sample, probes were hybridized and then ligated. For half of the sample, ligation was combined with HhaI (R6441, Promega) digestion. Agarose gel electrophoresis was used to check MLPA efficiency. PCR was performed, and data were quantified with GeneMarker software (version 1.5, Soft Genetics). The difference in the efficiency of the PCR for the individual samples was normalized by dividing the peak value of each probe by the peak of the control probes. CpGenome Universal Methylated DNA and Unmethylated DNA (Chemicon, Millipore) were included as controls. The methylation ratio was then calculated by dividing each normalized peak value of the digested sample by that of the corresponding undigested sample. The methylation ratio corresponded to the percentage of methylated sequences. A methylation ratio >0.25 was considered as “methylated”, which was consistent with a previous study [10 (link)].