Table 1.

Current methods for evaluating MGMT status

Parameter evaluated	Method	Description	Reference
Promoter methylation	Non-quantitative methylation-specific polymerase chain reaction (MSP)	DNA is treated with bisulfite, which converts unmethylated cytosine to uracil without modifying 5-methylcytosine. Using primers that are specific to methylated or unmethylated sequences, PCR is performed and analyzed by gel electrophoresis to provide qualitative results	[4, 6]
	Quantitative methylation-specific PCR (qMSP)	qMSP is similar to the non-quantitative MSP assay but provides quantitative results after normalization to an unmethylated gene	[6, 40]
	Pyrosequencing	A method of DNA sequencing, also based on bisulfite conversion and PCR, offering a quantitative determination of methylation of each individual CpG site sequenced	[13]
	Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MPLA)	Uses methylation-sensitive restriction enzymes to give semi-quantitative results for methylation status	[4, 13]
	Infinium Methylation EPIC BeadChip Array	Genome-wide methylation profiling of 850,000 CpG sites, including the MGMT genomic region	[6]
mRNA expression	Quantitative real-time polymerase chain reaction (qRT-PCR)	Measurement of MGMT transcript expression	[13, 43]
Protein expression	Immunohistochemistry	Tumor cells with nuclear staining are counted as MGMT-positive, and the percentage of positive cells is assessed to determine MGMT status	[13, 50]
Protein activity	Enzymatic assays	Sample is incubated with 3H-labeled O6 methyl-guanine, and the transfer of 3H-labeled methyl groups to the MGMT protein is measured by isolation of the 3H-labeled MGMT molecules	[4, 40]