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Cancer Gene Profiling

Volume 576 of the series Methods in Molecular Biology pp 155-170

Date:

Quantitative DNA Methylation Profiling on a High-Density Oligonucleotide Microarray

  • Anne FassbenderAffiliated withScience Department, Epigenomics AG
  • , Jörn LewinAffiliated withScience Department, Epigenomics AG
  • , Thomas KönigAffiliated withScience Department, Epigenomics AG
  • , Tamas RujanAffiliated withScience Department, Epigenomics AG
  • , Cecile PeletAffiliated withScience Department, Epigenomics AG
  • , Ralf LescheAffiliated withScience Department, Epigenomics AG
  • , Jürgen DistlerAffiliated withScience Department, Epigenomics AG
  • , Matthias SchusterAffiliated withScience Department, Epigenomics AG

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Summary

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.

Key words

DNA methylation DMH Microarray Normalization Calibration