DNA was extracted from the exfoliated EUCs of 46 subjects using the QIAamp DNA Blood Mini Kit (Qiagen, Valenica, CA) according to manufacturer’s instruction. Enrichment of CpG-methylated DNA was performed using the MethylMiner™ Methylated DNA Enrichment Kit (Invitrogen/Life Technologies, Grand Island, NY) and 250 µg of DNA. Enriched DNA was amplified and hybridized to Affymetrix Human Promoter 1.0R arrays (Affymetrix, Santa Clara, CA) as previously described.11 (link) The Affymetrix Human Promoter 1.0R arrays represent >25,500 human promoter regions, ~13,000 of which contain CpG islands, known targets of DNA methylation. Data were normalized using robust multichip average29 (link) and bioinformatically summarized at the CpG island level based on the Human Genome 18 (HG18) assembly. Average methylation abundance levels mapped to gene promoter regions were used in this analysis, as previously defined.11 (link) Specifically, in order to calculate the average methylation abundance for each gene, the methylation abundance levels for all CpG sites mapped to the same promoter region were summed and then divided by the number of CpG sites for that gene. Microarray data have been submitted to National Center for Biotechnology Information (NCBI) Gene Expression Omnibus repository30 (link) and are available under accession number GSE58499 (www.ncbi.nlm.nih.gov/geo).
Rager J.E., Miller S., Tulenko S.E., Smeester L., Ray P.D., Yosim A., Currier J.M., Ishida M.C., González-Horta M.D., Sánchez-Ramírez B., Ballinas-Casarrubias L., Gutiérrez-Torres D.S., Drobná Z., Del Razo L.M., García-Vargas G.G., Kim W.Y., Zhou Y.H., Wright F.A., Stýblo M, & Fry R.C. (2015). Identification of Novel Gene Targets and Putative Regulators of Arsenic-Associated DNA Methylation in Human Urothelial Cells and Bladder Cancer. Chemical research in toxicology, 28(6), 1144-1155.
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