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||**
From the Departments of Pathology,
* Medicine,
and Radiation Oncology,
|| Brigham and Women’s Hospital, Boston, Massachusetts; the Departments of Medical Oncology
and Radiation Oncology,
** Dana-Farber Cancer Institute, Boston, Massachusetts; Harvard Medical School,
Boston, Massachusetts; the Departments of Epidemiology and Biostatistics,
¶ Harvard School of Public Health, Boston, Massachusetts; and the Departments of Surgery and of Biochemistry and Molecular Biology,
University of Southern California Keck School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, California
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. However, no study has comprehensively evaluated the precision and performance characteristics of sodium bisulfite conversion and subsequent quantitative methylation assay. 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. To measure the precision of bisulfite conversion, we bisulfite-treated seven different aliquots of DNA from each of four paraffin-embedded colon cancer samples. To assess run-to-run variation, we repeated MethyLight five times. 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). Interclass correlation coefficients were 0.74 to 0.84 for the three loci, indicating good reproducibility. DNA mixing study with methylated and unmethylated DNA showed good linearity of the assay. Of 272 colorectal cancers evaluated, most showed PMR either <1 or >10, and promoter methylation (PMR >4) was tightly associated with loss of respective protein expression (P < 10–16). In conclusion, sodium bisulfite conversion and quantitative MethyLight assays have good precision and linearity and can be effectively used for high-throughput DNA methylation analysis on paraffin-embedded tissue.
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