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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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2017-04-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114533 |
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| author | Gu Zhou Craig Parfett Cathy Cummings-Lorbetskie Gong-Hua Xiao Daniel Desaulniers |
| author_facet | Gu Zhou Craig Parfett Cathy Cummings-Lorbetskie Gong-Hua Xiao Daniel Desaulniers |
| author_sort | Gu Zhou |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-c6d6f80b91ab4edc8eefb5efe2ddd1ab |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2017-04-01 |
| publisher | Taylor & Francis Group |
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| series | BioTechniques |
| spelling | doaj-art-c6d6f80b91ab4edc8eefb5efe2ddd1ab2025-08-20T02:26:03ZengTaylor & Francis GroupBioTechniques0736-62051940-98182017-04-0162415716410.2144/000114533Two-color fluorescent cytosine extension assay for the determination of global DNA methylationGu Zhou0Craig Parfett1Cathy Cummings-Lorbetskie2Gong-Hua Xiao3Daniel Desaulniers41Mechanistic Studies Division, Environmental Health Science and Research Bureau, HECSB, Health Canada, Ottawa, Canada1Mechanistic Studies Division, Environmental Health Science and Research Bureau, HECSB, Health Canada, Ottawa, Canada2Hazard Identification Division, Environmental Health Science and Research Bureau HECSB, Health Canada, Ottawa, Canada2Hazard Identification Division, Environmental Health Science and Research Bureau HECSB, Health Canada, Ottawa, Canada2Hazard Identification Division, Environmental Health Science and Research Bureau HECSB, Health Canada, Ottawa, CanadaHere, 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.https://www.future-science.com/doi/10.2144/000114533methylation sensitive restriction enzymedual labelaccuracyprecision |
| spellingShingle | Gu Zhou Craig Parfett Cathy Cummings-Lorbetskie Gong-Hua Xiao Daniel Desaulniers Two-color fluorescent cytosine extension assay for the determination of global DNA methylation BioTechniques methylation sensitive restriction enzyme dual label accuracy precision |
| title | Two-color fluorescent cytosine extension assay for the determination of global DNA methylation |
| title_full | Two-color fluorescent cytosine extension assay for the determination of global DNA methylation |
| title_fullStr | Two-color fluorescent cytosine extension assay for the determination of global DNA methylation |
| title_full_unstemmed | Two-color fluorescent cytosine extension assay for the determination of global DNA methylation |
| title_short | Two-color fluorescent cytosine extension assay for the determination of global DNA methylation |
| title_sort | two color fluorescent cytosine extension assay for the determination of global dna methylation |
| topic | methylation sensitive restriction enzyme dual label accuracy precision |
| url | https://www.future-science.com/doi/10.2144/000114533 |
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