Psq 96ma system

For 73/104 (70%) tumor DNA samples, MGMT methylation was analyzed using the PyroMark MGMT kit (Qiagen). Chemically methylated and unmethylated human genomic DNA controls (EpiTect PCR Control DNA Set, Qiagen) were included in each batch. In brief, 40 ng of tumor DNAs were extracted from paraffin-embedded tissue blocks using the FFPE DNA Extraction Kit (Zymo Research, Orange, CA). DNAs were then bisulfite-modified using the EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer’s recommendations. The CpG pyrosequencing methylation assay using the Qiagen kit was performed on a PSQ 96 MA system (Qiagen) according to the manufacturer’s protocol. The PyroMark MGMT kit quantifies the level of methylation at five individual CpG sites within exon 1 of MGMT using the Pyromark CpG software (Qiagen). The MGMT promoter was defined as unmethylated when the mean methylation of the five CpG sites was <8%, and methylated when this value was ≥ 8% [26 ].

To determine promoter methylation, 500 ng tumor DNA were biosulfite-converted, amplified by the PyroMark MGMT kit (Qiagen) followed by pyrosequencing analysis. Five potentially methylated sites in the first exon (+17 to +39) of the MGMT gene were analyzed by the EZ DNA Methylation-Gold^TM Kit (Zymo Research) following the manufacturer’s suggestions. The methylation level was analyzed on the PSQ96 MA system (Qiagen) and quantified by the Qiagen Pyro-Mark CpG software (Qiagen). Hyper-methylation is defined by the average methylation sites equal or higher than 10% on the 5 CpG, otherwise regarded as hypo-methylation.

DNA methylation was analyzed using bisulfite pyrosequencing as described previously [37 (link),38 (link)]. Briefly, genomic DNA (1 μg) was modified with sodium bisulfite using an EpiTect Bisulfite kit (Qiagen). Pyrosequencing was then carried out using a PSQ 96MA system (Qiagen) with a Pyro Gold Reagent kit (Qiagen), and the results were analyzed using Pyro Q-CpG software (Qiagen). A cutoff value of 15% was used to define genes as methylation-positive. Using five classic CIMP markers (MINT1, MINT2, MINT12, MINT31 and MLH1) and CDKN2A (p16), tumors were defined as CIMP-positive (three or more loci showed methylation) or CIMP-high (CIMP-H, four or more loci showed methylation). Methylation of SMOC1, GALNT14, SFRP1, SFRP2, IGFBP7, SOX5 and long interspersed nucleotide element 1 (LINE-1) was also analyzed using bisulfite pyrosequencing. The primer sequences used were as previously reported [9 (link),31 (link),39 (link)].