Table 3.

Comparison of established protocols with the present study protocols in MGMT methylation analysis

Assay	Target CpGs	Strengths	Weaknesses	References
MSP (qMSP)	DMR2 (CpG 71–86, 76–87)	Simple Low cost	Unable to analyze heterogeneous methylation	[12, 17]
PSQ	DMR2 (CpG 74–78)	High accuracy	High cost Limitation of Amplicon length	[12, 23]
MS-HRM	DMR2 (CpG 72–83, 72–89, 84–89)	Capable of analyzing heterogeneous methylation	Low accuracy Limitation of Amplicon length	[10, 12, 22]
HPLC	500 bp upstream of TSS (CpG 1–20) DMR1 (CpG 22–59) DMR2 (CpG 72–98)	Simple Low cost Capable of analyzing long amplicons	Unstable measurement of paraffin-embedded specimen	Present study

CpG numbers on MGMT CpG islands were defined by Harris et al. [16]

DMR differentially methylated region, MSP methylation-specific polymerase chain reaction, PSQ pyrosequencing,

qMSP quantitative MSP, MS-HRM methylation-sensitive high-resolution melting analysis, TSS transcription start site