Pyromark vacuum workstation

The converted DNA was then amplified in a MGMT promoter specific PCR using the PyroMark Q24 System from Qiagen GmbH (Hilden, Germany). By adding 3 μL of converted DNA we followed the manufacturer instruction, but adapted the PCR cycling protocol (S1 Table).
After amplification, we followed the manufracturer instructions by using the PyroMark Vacuum Workstation and PyroMark Q24 System from Qiagen GmbH (Hilden, Germany) to detect methylated CpG islands in MGMT promoter DNA.
Results were determined by PyroMark Q24 software Qiagen GmbH (Hilden, Germany). Five CpG islands were measured and results reported as percentages. Out of five percent values we calculated a binary (MGMTbi) and a continuous (MGMTco) variable. For each CpG island the percentage of methylation was measured. In addition, the mean percentage of methylation across all CpG islands was calculated for each tumor. MGMT methylation was defined as positive if this mean value was >10%.
The continuous variable MGMTco (0–5) considered every single CpG island. The MGMTco value describes the number of CpG islands with a methylation of >10%.

The Pyromark Q24 Advanced Reagent kit (Qiagen, Manchester, UK) was used for preparation of pyrosequencing reactions following the manufacturer’s instructions, using the PyroMark vacuum workstation and PyroMark Q24 sequencer (Qiagen, Manchester, UK). One positive control and one negative control were included on each plate to act as inter-plate controls. The designed assay sequences for analysis (Table 1) were programmed into the PyroMark Q24 software (v2.0., Qiagen, Manchester, UK), and the assays were run according to machine-default settings. Output data included a calculated methylation percentage at each CpG site in the sequences analysed, with the average methylation of all sites being calculated for statistical analysis of a given candidate gene.