The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status
DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposu...
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| Format: | Article |
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Wiley Blackwell
2018
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| Online Access: | http://hdl.handle.net/1721.1/117649 https://orcid.org/0000-0002-5472-3621 https://orcid.org/0000-0003-4322-3573 |
| _version_ | 1811089539019571200 |
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| author | Townsend, Todd A. Manjanatha, Mugimane G. Parrish, Marcus Curtis Engelward, Bevin P |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Townsend, Todd A. Manjanatha, Mugimane G. Parrish, Marcus Curtis Engelward, Bevin P |
| author_sort | Townsend, Todd A. |
| collection | MIT |
| description | DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, “EpiComet,” which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. |
| first_indexed | 2024-09-23T14:20:47Z |
| format | Article |
| id | mit-1721.1/117649 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T14:20:47Z |
| publishDate | 2018 |
| publisher | Wiley Blackwell |
| record_format | dspace |
| spelling | mit-1721.1/1176492022-09-29T08:51:14Z The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status Townsend, Todd A. Manjanatha, Mugimane G. Parrish, Marcus Curtis Engelward, Bevin P Massachusetts Institute of Technology. Department of Biological Engineering Parrish, Marcus Curtis Engelward, Bevin P DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, “EpiComet,” which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. 2018-09-06T17:44:36Z 2018-09-06T17:44:36Z 2017-07 2018-08-28T17:55:07Z Article http://purl.org/eprint/type/JournalArticle 0893-6692 1098-2280 http://hdl.handle.net/1721.1/117649 Townsend, Todd A. et al. “The Development and Validation of EpiComet-Chip, a Modified High-Throughput Comet Assay for the Assessment of DNA Methylation Status.” Environmental and Molecular Mutagenesis 58, 7 (July 2017): 508–521 © 2017 Wiley Periodicals, Inc https://orcid.org/0000-0002-5472-3621 https://orcid.org/0000-0003-4322-3573 http://dx.doi.org/10.1002/EM.22101 Environmental and Molecular Mutagenesis Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Wiley Blackwell PMC |
| spellingShingle | Townsend, Todd A. Manjanatha, Mugimane G. Parrish, Marcus Curtis Engelward, Bevin P The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status |
| title | The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status |
| title_full | The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status |
| title_fullStr | The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status |
| title_full_unstemmed | The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status |
| title_short | The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status |
| title_sort | development and validation of epicomet chip a modified high throughput comet assay for the assessment of dna methylation status |
| url | http://hdl.handle.net/1721.1/117649 https://orcid.org/0000-0002-5472-3621 https://orcid.org/0000-0003-4322-3573 |
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