CpGenome Universal Methylated DNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Aliquote in small quantity to avoid freeze-thaw cycles and Storage at -70°C for long-term use (2-3 yrs)

Upstream tips
Aliquote in small quantity to avoid freeze-thaw cycles and Storage at -70°C for long-term use (2-3 yrs)

Publication protocol

Quantitative and Allelic Methylation Analyses by qMSP Followed by Pyrosequencing
Quantitative and allelic methylation analyses were performed using qMSP and melting analyses followed by pyrosequencing of methylation-positive samples being heterozygous for the rs16906252 SNP (). The genomic location of the primers is shown in . For normalization purposes, we used an assay based on Alu sequences depleted of CpG sites by evolutionary deamination (). This assay is less susceptible to normalization errors caused by aneuploidy and copy number changes often observed in cancer cells. Additional details are in the .

Quantification of Methylation Levels Using qMSP
Methylation levels of the samples were estimated relative to universal methylated DNA (Chemicon, Millipore, Billerica, MA) using the 2 quantification approach (), in which ΔΔCt = glioblastoma sample (Ct – Ct) − universal methylated DNA (Ct − Ct) (). This approach implies that the assays have approximately the same PCR efficiency () (). For the assay, = 2.0; for the Alu assay, = 1.9. Samples were analyzed in duplicate and the average Ct values were used in the calculations. Only samples for which both replicates amplified in the assay having a melting profile resembling the melting profile of universal methylated DNA were considered methylation positive. Samples with a mean Ct value above 17 in the Alu assay were bisulfite converted again and repeated. Samples with methylation levels below 0.1% were analyzed as unmethylated. This technical cutoff was defined following an evaluation of a serial dilution series of methylated DNA into unmethylated. Samples with a methylation level below 5% were scored as low-level methylation; samples with a methylation level between 5% and 20% as medium; and samples with a methylation level above 20% as high. No template control and unmethylated DNA, prepared as described (), were included as negative controls.

Standard Bisulfite Pyrosequencing
PCR and pyrosequencing were performed using the Therascreen (R) MGMT Pyro (R) kit according to the manufacturer’s instructions with slight modifications (). The CpG sites analyzed by the standard pyrosequencing assay are shown in . Samples with a mean methylation level below 10% were considered unmethylated. Samples with a mean methylation level between 10% and 25% were scored as low-level methylation; samples with a mean methylation level between 25% and 50% as medium; and samples with a mean methylation level above 50% as high.

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