Micropatterned comet assay enables high throughput and sensitive DNA damage quantification
The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Oxford University Press
2016
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| Online Access: | http://hdl.handle.net/1721.1/100735 |
| _version_ | 1826215888227926016 |
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| author | Engelward, B. P. Ge, Jing Chow, Danielle N. Fessler, Jessica L. Weingeist, David M. Wood, David K. Engelward, Bevin P. |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Engelward, B. P. Ge, Jing Chow, Danielle N. Fessler, Jessica L. Weingeist, David M. Wood, David K. Engelward, Bevin P. |
| author_sort | Engelward, B. P. |
| collection | MIT |
| description | The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H[subscript 2]O[subscript 2]-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. |
| first_indexed | 2024-09-23T16:38:38Z |
| format | Article |
| id | mit-1721.1/100735 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T16:38:38Z |
| publishDate | 2016 |
| publisher | Oxford University Press |
| record_format | dspace |
| spelling | mit-1721.1/1007352022-10-03T07:22:25Z Micropatterned comet assay enables high throughput and sensitive DNA damage quantification Engelward, B. P. Ge, Jing Chow, Danielle N. Fessler, Jessica L. Weingeist, David M. Wood, David K. Engelward, Bevin P. Massachusetts Institute of Technology. Department of Biological Engineering Engelward, Bevin P. Ge, Jing Chow, Danielle N. Fessler, Jessica L. Weingeist, David M. Engelward, Bevin P. The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H[subscript 2]O[subscript 2]-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. National Institute of Environmental Health Sciences (Grant U01-ES016-45) National Institute of Environmental Health Sciences (Grant R43-ES021116-01) National Institute of Environmental Health Sciences (Training Grant T32-ES0702) Massachusetts Institute of Technology. Undergraduate Research Opportunities Program 2016-01-07T01:05:39Z 2016-01-07T01:05:39Z 2014-12 2014-09 Article http://purl.org/eprint/type/JournalArticle 0267-8357 1464-3804 http://hdl.handle.net/1721.1/100735 Ge, J., D. N. Chow, J. L. Fessler, D. M. Weingeist, D. K. Wood, and B. P. Engelward. “Micropatterned Comet Assay Enables High Throughput and Sensitive DNA Damage Quantification.” Mutagenesis 30, no. 1 (December 19, 2014): 11–19. en_US http://dx.doi.org/10.1093/mutage/geu063 Mutagenesis Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Oxford University Press Prof. Engelward via Howard Silver |
| spellingShingle | Engelward, B. P. Ge, Jing Chow, Danielle N. Fessler, Jessica L. Weingeist, David M. Wood, David K. Engelward, Bevin P. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification |
| title | Micropatterned comet assay enables high throughput and sensitive DNA damage quantification |
| title_full | Micropatterned comet assay enables high throughput and sensitive DNA damage quantification |
| title_fullStr | Micropatterned comet assay enables high throughput and sensitive DNA damage quantification |
| title_full_unstemmed | Micropatterned comet assay enables high throughput and sensitive DNA damage quantification |
| title_short | Micropatterned comet assay enables high throughput and sensitive DNA damage quantification |
| title_sort | micropatterned comet assay enables high throughput and sensitive dna damage quantification |
| url | http://hdl.handle.net/1721.1/100735 |
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