Quantitative DNA methylation profiling on a high-density oligonucleotide microarray

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 576; p. 155
• Main Authors: Fassbender, Anne, Lewin, Jörn, König, Thomas, Rujan, Tamas, Pelet, Cecile, Lesche, Ralf, Distler, Jürgen, Schuster, Matthias
• Format: Journal Article
• Language: English
• Published: United States 01.01.2010
• Subjects: 5' Untranslated Regions; Calibration; CpG Islands; DNA Fragmentation; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome; Humans; Molecular Biology - methods; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotides - genetics; Polymerase Chain Reaction
• ISSN: 1940-6029
• 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.