Fassbender Anne, Lewin Jörn, König Thomas, Rujan Tamas, Pelet Cecile, Lesche Ralf, Distler Jürgen, Schuster Matthias
Science Department, Epigenomics AG, Berlin, Germany.
Methods Mol Biol. 2010;576:155-70. doi: 10.1007/978-1-59745-545-9_9.
Recently, the analysis and functional elucidation of CpG island methylation has become a focus area of genomic research. Deviations from the normal parental imprinting pattern have been shown to cause developmental defects associated with serious symptoms. Aberrant DNA methylation of tumor suppressor and other functional genes, especially when found in 5' untranslated regions and early exons, has been associated with tumorigenesis. In the context of applying DNA methylation analysis for the molecular characterization of cancer and other diseases, standardized protocols enabling parallel genome-wide methylation profiling of numerous samples are required. DNA methylation profiling is described using a CpG island microarray representing more than 50,000 CpG-rich DNA fragments. Fragments were selected to represent the vast majority of known 5'-untranslated regions as well as the first exons of thousands of genes. Measurement probes were designed to represent these fragments were displayed on an Affymetrix custom array. A modified procedure for differential methylation hybridization (DMH) is described for methylation enrichment. Application of a novel signal normalization concept enables accurate and reproducible measurements using a single fluorescence channel. The use of defined calibrator material allows quantification of DNA methylation patterns by DMH in a massively parallel fashion.
最近，CpG岛甲基化的分析和功能阐释已成为基因组研究的一个重点领域。已表明与正常亲本印记模式的偏差会导致与严重症状相关的发育缺陷。肿瘤抑制基因和其他功能基因的异常DNA甲基化，特别是当在5'非翻译区和早期外显子中发现时，与肿瘤发生有关。在将DNA甲基化分析应用于癌症和其他疾病的分子特征研究时，需要能够对大量样本进行全基因组甲基化平行分析的标准化方案。使用代表超过50,000个富含CpG的DNA片段的CpG岛微阵列来描述DNA甲基化分析。选择片段以代表绝大多数已知的5'-非翻译区以及数千个基因的第一个外显子。设计用于代表这些片段的测量探针显示在Affymetrix定制阵列上。描述了一种用于甲基化富集的差异甲基化杂交（DMH）的改进程序。应用一种新颖的信号归一化概念能够使用单个荧光通道进行准确且可重复的测量。使用定义的校准材料允许通过DMH以大规模平行方式对DNA甲基化模式进行定量。
