Published online 27 May 2009 Nucleic Acids Research, 2009, Vol. 37, No. 12 e89 doi:10.1093/nar/gkp413 Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation Sitharthan Kamalakaran 1 , Jude Kendall2, Xiaoyue Zhao 2 , Chunlao Tang 2 , Sohail Khan 2 , Kandasamy Ravi2, Theresa Auletta2, Michael Riggs 2 , Yun Wang 3 , A˚ slaug Helland3,4, Bjørn Naume 4 , Nevenka Dimitrova 1 , Anne-Lise Børresen-Dale3,5, Jim Hicks2 and Robert Lucito2,* 1Philips Research North America, Briarcliff Manor, NY 10510, USA, 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, 3Department of Genetics, Institute for Cancer Research, 4Department of Oncology, Norwegian Radium Hospital, Rikshospitalet University Hospital and 5Faculty Division, The Norwegian Radium Hospital Faculty of Medicine, University of Oslo, Oslo, Norway Received October 9, 2008; Revised April 29, 2009; Accepted May 2, 2009 ABSTRACT Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands cor- relates to genomic instability. This epigenetic mod- ification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methyla- tion status of over 25 000 CpG islands in the human genome. Experiments were performed to demon- strate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accu- racy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately iden- tifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species. INTRODUCTION It has become increasingly clear how epigenetic modiﬁca- tion can aﬀect the structure and the expression of genes encoded in the DNA. One such modiﬁcation is the methy- lation of cytosines that are 50 to guanines, so-called CpG dinucleotides. Found scattered across the genome, although at a lower than expected frequency, CpG dinu- cleotides also cluster into what have been termed CpG islands. The deﬁnition of a CpG islands diﬀers somewhat based on the algorithm used for identiﬁcation, two com- monly used algorithms being Gardiner-Garden and Frommer (1) and Takai-Jones (2). The islands identiﬁed can be classiﬁed as falling into two distinct classes, those that are overlapping or proximal (within 2000 bp) to the transcription start site (TSS) of genes and those that are not associated with any transcription start site (non-TSS) for an obvious gene. Most CpG islands proximal to the TSS of genes (TSS–CGIs) are largely unmethylated nor- mally, and methylation of these islands, as can occur during tumorigenesis, has been shown to correlate highly to the suppression of transcription (3). Of the non-TSS CpG islands (non-TSS–CGIs) in the genome, many of these are proximal or inclusive to repetitive sequences, and are generally heavily methylated in normal tissue (4,5). However, during tumorigenesis hypomethylation occurs at these islands (4,5), which can result in the expres- sion of certain repeats (6,7). Interestingly, this hypomethy- lation correlates to the severity of some cancers (8,9) and DNA breakage and genome instability (10). Under certain circumstances, which can occur in pathologies such as cancer, imprinting, development, tissue speciﬁcity and X-chromosome inactivation, TSS– CGIs can be heavily methylated (11). Speciﬁcally, in cancer, methylation of islands proximal to tumor suppres- sor genes such as p16, RASSF1A, BRCA1, is a frequent event (12–14). Since the analysis of such genes was pre- viously done one at a time using bisulﬁte sequencing, the value of accurate high-throughput methods is obvious. Several higher throughput methods, many array based, have been developed to identify CpG methylation in the *To whom correspondence should be addressed. Tel: +1 516 422 4138; Fax: +1 516 422 4109; Email: lucito@cshl.edu 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.