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Biotechniques. 2017 Apr 1;62(4):157-164. doi: 10.2144/000114533.

Two-color fluorescent cytosine extension assay for the determination of global DNA methylation.

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Mechanistic Studies Division, Environmental Health Science and Research Bureau, HECSB, Health Canada, Ottawa, Canada.
Hazard Identification Division, Environmental Health Science and Research Bureau HECSB, Health Canada, Ottawa, Canada.


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.


accuracy; dual label; methylation sensitive restriction enzyme; precision

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