title: Two-color fluorescent cytosine extension assay for the determination of global DNA methylation.
abstract: Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.
authors.affiliation: Mechanistic Studies Division, Environmental Health Science and Research Bureau, HECSB, Health Canada, Ottawa, Canada.
authors.forename: Gu
authors.initials: G
authors.lastname: Zhou
authors.affiliation: Mechanistic Studies Division, Environmental Health Science and Research Bureau, HECSB, Health Canada, Ottawa, Canada.
authors.forename: Craig
authors.initials: C
authors.lastname: Parfett
authors.affiliation: Hazard Identification Division, Environmental Health Science and Research Bureau HECSB, Health Canada, Ottawa, Canada.
authors.forename: Cathy
authors.initials: C
authors.lastname: Cummings-Lorbetskie
authors.affiliation: Hazard Identification Division, Environmental Health Science and Research Bureau HECSB, Health Canada, Ottawa, Canada.
authors.forename: Gong-Hua
authors.initials: GH
authors.lastname: Xiao
authors.affiliation: Hazard Identification Division, Environmental Health Science and Research Bureau HECSB, Health Canada, Ottawa, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Desaulniers
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000737:chemistry; D001681:Biological Assay / Q000379:methods*; D003596:Cytosine; D019175:DNA Methylation / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics; D000077209:Decitabine; D005453:Fluorescence; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D061251:Primary Cell Culture
chemical_list: D000077209:Decitabine; D003596:Cytosine; D004262:DNA Restriction Enzymes; D001374:Azacitidine
keywords: accuracy; dual label; methylation sensitive restriction enzyme; precision