![]() Of 272 colorectal cancers evaluated, most showed PMR either 10, and promoter methylation (PMR >4) was tightly associated with loss of respective protein expression ( P < 10 −16). DNA mixing study with methylated and unmethylated DNA showed good linearity of the assay. ![]() Interclass correlation coefficients were 0.74 to 0.84 for the three loci, indicating good reproducibility. Bisulfite-to-bisulfite coefficient of variation (CV) of PMR ranged from 0.10 to 0.38 (mean, 0.21), and run-to-run CV of PMR ranged from 0.046 to 0.60 (mean, 0.31). To assess run-to-run variation, we repeated MethyLight five times. To measure the precision of bisulfite conversion, we bisulfite-treated seven different aliquots of DNA from each of four paraffin-embedded colon cancer samples. We developed quantitative real-time polymerase chain reaction (MethyLight) to measure percentage of methylated reference (PMR, ie, degree of methylation) for the MGMT, MLH1, and CDKN2A (p16) promoters. However, no study has comprehensively evaluated the precision and performance characteristics of sodium bisulfite conversion and subsequent quantitative methylation assay. Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite.
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