Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis
作者： Shuji Ogino , Takako Kawasaki , Mohan Brahmandam , Mami Cantor , Gregory J. Kirkner
DOI: 10.2353/JMOLDX.2006.050135
关键词:
摘要: Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, typically rely on conversion of unmethylated cytosine uracil by sodium bisulfite. However, no study has comprehensively evaluated the precision performance characteristics bisulfite subsequent quantitative methylation assay. We developed real-time polymerase chain reaction (MethyLight) percentage methylated reference (PMR, ie, degree methylation) for MGMT, MLH1, CDKN2A (pl6) promoters. To conversion, we bisulfite-treated seven different aliquots from each four paraffin-embedded colon cancer samples. assess run-to-run variation, repeated MethyLight five times. Bisulfite-to-bisulfite coefficient variation (CV) PMR ranged 0.10 0.38 (mean, 0.21), CV 0.046 0.60 0.31). Interclass correlation coefficients were 0.74 0.84 three loci, indicating good reproducibility. mixing with showed linearity Of 272 colorectal cancers evaluated, most either 10, promoter (PMR >4) was tightly associated loss respective protein expression (P
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