Ogino Shuji, Kawasaki Takako, Brahmandam Mohan, Cantor Mami, Kirkner Gregory J, Spiegelman Donna, Makrigiorgos G Mike, Weisenberger Daniel J, Laird Peter W, Loda Massimo, Fuchs Charles S
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, USA.
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.
用于测量DNA甲基化的检测方法在表观遗传学研究和临床诊断中至关重要，通常依赖于亚硫酸氢钠将未甲基化的胞嘧啶转化为尿嘧啶。然而，尚无研究全面评估亚硫酸氢钠转化及后续定量甲基化检测的精密度和性能特征。我们开发了定量实时聚合酶链反应（MethyLight）来测量MGMT、MLH1和CDKN2A（p16）启动子的甲基化参考百分比（PMR，即甲基化程度）。为了测量亚硫酸氢盐转化的精密度，我们对来自四个石蜡包埋结肠癌样本的每份DNA进行了七次不同的亚硫酸氢盐处理。为了评估批间差异，我们重复进行了五次MethyLight检测。PMR的亚硫酸氢盐间变异系数（CV）范围为0.10至0.38（平均值为0.21），PMR的批间CV范围为0.046至0.60（平均值为0.31）。三个位点的组内相关系数为0.74至0.84，表明具有良好的重复性。甲基化和未甲基化DNA的DNA混合研究显示该检测具有良好的线性。在评估的272例结直肠癌中大部分PMR<1或PMR>10，启动子甲基化（PMR>4）与相应蛋白表达缺失紧密相关（P<10-16）。总之，亚硫酸氢钠转化和定量MethyLight检测具有良好的精密度和线性，可有效用于石蜡包埋组织的高通量DNA甲基化分析。
