Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis
Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis
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DOI：
10.2353/jmoldx.2006.050135
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发表时间：
2006-05-01
影响因子：
4.1
通讯作者：
Fuchs, Charles S.
中科院分区：
文献类型：
--
作者：
Ogino, Shuji;Kawasaki, Takako;Fuchs, Charles S.
在表观遗传学研究和临床诊断中重要的测量DNA甲基化的测定通常依赖于未甲基化的转化。胞嘧啶通过亚硫酸氢钠转化为尿嘧啶。然而，没有研究全面评价亚硫酸氢钠转化和随后的定量甲基化测定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、MLH 1和CDKN 2A（p16）启动子的甲基化参考（PMR，即甲基化程度）百分比。为了测量亚硫酸氢盐转化的精确度，我们对来自四个石蜡包埋的结肠癌样本中的每个的七个不同的DNA等分试样进行亚硫酸氢盐处理。为了评估运行间差异，我们重复MethyLight五次。PMR的亚硫酸氢盐-亚硫酸氢盐变异系数（CV）范围为0.10 - 0.38（平均值为0.21），PMR的运行间CV范围为0.046 - 0.60（平均值为0.31）。3个基因座的组间相关系数为0.74 ~ 0.84，重复性好。甲基化和未甲基化的DNA混合研究。DNA检测结果线性良好。在272例结直肠癌中，大多数显示PMR> < 1 or >10，启动子甲基化（PMR > 4）与相应蛋白表达缺失密切相关（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。
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.