Two-color fluorescent cytosine extension assay for the determination of global DNA methylation
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 diges...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2017-04-01
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| Series: | BioTechniques |
| Subjects: | |
| Online Access: | https://www.future-science.com/doi/10.2144/000114533 |
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| Summary: | 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. |
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| ISSN: | 0736-6205 1940-9818 |