Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis

J Mol Diagn. 2006 May;8(2):209-17. doi: 10.2353/jmoldx.2006.050135.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins / genetics
  • Collagen Type II / genetics
  • Colorectal Neoplasms / genetics
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics
  • DNA / metabolism*
  • DNA Methylation
  • Gene Expression
  • Gene Expression Regulation, Neoplastic
  • Humans
  • MutL Protein Homolog 1
  • Nuclear Proteins / genetics
  • Polymerase Chain Reaction / methods*
  • Sulfites / analysis*
  • Time Factors


  • Adaptor Proteins, Signal Transducing
  • COL2A1 protein, human
  • Carrier Proteins
  • Collagen Type II
  • Cyclin-Dependent Kinase Inhibitor p16
  • MLH1 protein, human
  • Nuclear Proteins
  • Sulfites
  • DNA
  • MutL Protein Homolog 1