Determining methylation status of methylguanine DNA methyl transferase (MGMT) from formalin-fixed, paraffin embedded tumor tissue

O-6-methylguanine-DNA methyltransferase (MGMT) has been associated with resistance to alkylating agent cancer therapy in Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. Lower expression or silencing of the MGMT protein by promoter methylation has been reported to improve survival in patients with GBM [1]. This protocol describes bisulfite conversion, methylation sensitive PCR amplification and data analysis/interpretation.This protocol differs from published protocols in that it:
• Describes a detailed method to measure MGMT using DNA extracted from solid tumor tissue. We have optimized the DNA extraction by using FFPE tissue blocks that contain greater than 50% tumor tissue, when non-tumor tissue was also present. Performance of this assay is compromised when lower quantities of tumor cells are used as the methylation status of tumor cells is diluted out by methylation status of normal cells.
• The measurement of MGMT could be further (enhanced) optimized using a percentage of methylation ration cutoff of 2 as methylated.
• The machine specifications detailed here are specific to measuring MGMT from PPFE tumor tissue.

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