Pyromark q24 cpg mgmt kit

The histopathology was performed by an experienced neuropathologist (R.B.) and tissue samples classified according to the current WHO classification of tumors of the CNS. For defining the MGMT methylation status five CpG sites in the MGMT promoter were analyzed using the PyroMark MD System (Qiagen). Subsequent sample preparation and pyrosequencing was performed as described in the Pyro-Mark MD Sample Prep Guidelines using the PyroMark Q24 CpG MGMT kit (part number 970032 Qiagen).

The analysis was performed at the Molecular Pathology Laboratory at IRCCS Policlinico Sant’Orsola-Malpighi of Bologna. MGMT-promoter methylation status was evaluated using pyrosequencing. To be considered fully evaluable, the samples had to contain more than 80% tumor cells. DNA extraction from formalin-fixed, paraffin-embedded tissue (from surgical resection specimen or biopsy of primary tumor or metastases) was performed after deparaffinization using a purification kit (MasterPure DNA, Epicentre, Madison, WI, USA). Genomic DNA was modified by bisulfite conversion (EZ DNA Methylation Gold Kit, Zymo, Irvine, CA, USA). Pyrosequencing was performed using the PyroMark Q24 CpG MGMT kit (Qiagen, Hilden, Germany) on a PyroMark Q24 System (Qiagen). Data were analyzed and quantified with the PyroMark Q24 Software 2.0.7 (Qiagen). The mean percentage of the five CpG methylated islands detected by the kit was used for analysis. An 8% cut-off was used, accordingly to neuro-oncology clinical practice: MGMT was considered methylated if methylated alleles were more numerous than not-methylated alleles by at least 8%; otherwise MGMT was scored as not methylated [29 (link),30 (link)].