Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process
Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process
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DOI：
10.1186/s13148-016-0308-0
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发表时间：
2017-01-13
影响因子：
5.7
通讯作者：
Kang, Gyeong Hoon
中科院分区：
文献类型：
--
作者：
Wen, Xianyu;Jeong, Seorin;Kang, Gyeong Hoon
背景资料：福尔马林固定石蜡包埋（FFPE）组织是分析DNA甲基化变化和研究各种疾病的重要资源。然而，福尔马林固定引入链间交联，这可能导致未甲基化胞嘧啶的不完全亚硫酸氢盐转化，这可能导致焦磷酸测序测定中甲基化水平的测量值错误升高。长散布核苷酸元件-1（LINE-1）是DNA重复转座元件的主要组成部分，其甲基化与DNA整体甲基化密切相关。为了确定福尔马林固定是否可能影响LINE-1重复元件中甲基化的测量值，以及延长的DNA热诱导变性是否可能减少福尔马林固定引起的测量值的人为增加，我们使用焦磷酸测序法分析了配对的新鲜冷冻（FF）和FFPE异种移植组织样品的LINE-1甲基化水平。为了进一步确认加热步骤在LINE-1或单基因甲基化水平的测量中的效果，我们分析了胃癌和结直肠癌的FFPE组织样本的LINE-1和8个单基因的甲基化状态，分别为：福尔马林固定导致LINE-1甲基化的测量值增加，而不管固定的持续时间。发现在亚硫酸氢盐转化之前将DNA在95 ℃下延长加热30分钟（1）降低了配对FF和FFPE组织样品之间测量值的差异，（2）降低了胃癌的FFPE组织样品中LINE-1甲基化水平测量值的标准偏差，（3）提高FFPE检测结直肠癌组织单基因甲基化水平的性能。福尔马林固定导致LINE-1甲基化测量值的人为增加，并且DNA样品的延长加热的应用降低了配对FF和FFPE组织样品之间LINE-1甲基化的测量值的差异。DNA样品的延长加热的应用改进了FFPE组织样品中LINE-1或单基因甲基化水平的基于亚硫酸氢盐转化的测量。
Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively.Results: Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 degrees C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer.Conclusions: Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.