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A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage

The Comet or single-cell gel electrophoresis assay is a highly sensitive method to measure cellular, nuclear genome damage. However, low throughput can limit its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases thr...

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Autores principales: Li, Jianfeng, Beiser, Alison, Dey, Nupur B, Takeda, Shunichi, Saha, Liton Kumar, Hirota, Kouji, Parker, L Lynette, Carter, Mariah, Arrieta, Martha I, Sobol, Robert W
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469923/
https://www.ncbi.nlm.nih.gov/pubmed/36110898
http://dx.doi.org/10.1093/nargab/lqac065
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author Li, Jianfeng
Beiser, Alison
Dey, Nupur B
Takeda, Shunichi
Saha, Liton Kumar
Hirota, Kouji
Parker, L Lynette
Carter, Mariah
Arrieta, Martha I
Sobol, Robert W
author_facet Li, Jianfeng
Beiser, Alison
Dey, Nupur B
Takeda, Shunichi
Saha, Liton Kumar
Hirota, Kouji
Parker, L Lynette
Carter, Mariah
Arrieta, Martha I
Sobol, Robert W
author_sort Li, Jianfeng
collection PubMed
description The Comet or single-cell gel electrophoresis assay is a highly sensitive method to measure cellular, nuclear genome damage. However, low throughput can limit its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases throughput and reduces variation due to simultaneous processing and automated analysis of 96 samples. To advance throughput further, we developed a 384-well CometChip platform that allows analysis of ∼100 cells per well. The 384-well CometChip extends the capacity by 4-fold as compared to the 96-well system, enhancing application for larger DNA damage analysis studies. The overall sensitivity of the 384-well CometChip is consistent with that of the 96-well system, sensitive to genotoxin exposure and to loss of DNA repair capacity. We then applied the 384-well platform to screen a library of protein kinase inhibitors to probe each as enhancers of etoposide induced DNA damage. Here, we found that 3-methyladenine significantly increased levels of etoposide-induced DNA damage. Our results suggest that a 384-well CometChip is useful for large-scale DNA damage analyses, which may have increased potential in the evaluation of chemotherapy efficacy, compound library screens, population-based analyses of genome damage and evaluating the impact of environmental genotoxins on genome integrity.
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spelling pubmed-94699232022-09-14 A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage Li, Jianfeng Beiser, Alison Dey, Nupur B Takeda, Shunichi Saha, Liton Kumar Hirota, Kouji Parker, L Lynette Carter, Mariah Arrieta, Martha I Sobol, Robert W NAR Genom Bioinform High Throughput Sequencing Methods The Comet or single-cell gel electrophoresis assay is a highly sensitive method to measure cellular, nuclear genome damage. However, low throughput can limit its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases throughput and reduces variation due to simultaneous processing and automated analysis of 96 samples. To advance throughput further, we developed a 384-well CometChip platform that allows analysis of ∼100 cells per well. The 384-well CometChip extends the capacity by 4-fold as compared to the 96-well system, enhancing application for larger DNA damage analysis studies. The overall sensitivity of the 384-well CometChip is consistent with that of the 96-well system, sensitive to genotoxin exposure and to loss of DNA repair capacity. We then applied the 384-well platform to screen a library of protein kinase inhibitors to probe each as enhancers of etoposide induced DNA damage. Here, we found that 3-methyladenine significantly increased levels of etoposide-induced DNA damage. Our results suggest that a 384-well CometChip is useful for large-scale DNA damage analyses, which may have increased potential in the evaluation of chemotherapy efficacy, compound library screens, population-based analyses of genome damage and evaluating the impact of environmental genotoxins on genome integrity. Oxford University Press 2022-09-13 /pmc/articles/PMC9469923/ /pubmed/36110898 http://dx.doi.org/10.1093/nargab/lqac065 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle High Throughput Sequencing Methods
Li, Jianfeng
Beiser, Alison
Dey, Nupur B
Takeda, Shunichi
Saha, Liton Kumar
Hirota, Kouji
Parker, L Lynette
Carter, Mariah
Arrieta, Martha I
Sobol, Robert W
A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage
title A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage
title_full A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage
title_fullStr A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage
title_full_unstemmed A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage
title_short A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage
title_sort high-throughput 384-well cometchip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced dna damage
topic High Throughput Sequencing Methods
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469923/
https://www.ncbi.nlm.nih.gov/pubmed/36110898
http://dx.doi.org/10.1093/nargab/lqac065
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