A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage.
A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage.
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DOI：
10.1093/nargab/lqac065
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发表时间：
2022-09
影响因子：
4.6
通讯作者：
中科院分区：
文献类型：
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彗星或单细胞凝胶电泳测定是一种高度敏感的方法来测量细胞，核基因组损伤。然而，低通量限制了其在大规模研究中的应用。为了克服这些限制，最近开发了96口CometChip平台，通过同时处理和自动分析96个样品，提高了吞吐量，减少了变化。为了进一步提高吞吐量，我们开发了384孔CometChip平台，每孔可分析约100个细胞。384孔CometChip的容量是96孔系统的4倍，可用于更大规模的DNA损伤分析研究。384孔CometChip的总体灵敏度与96孔系统一致，对基因毒素暴露和DNA修复能力的丧失敏感。然后，我们应用384孔平台筛选蛋白激酶抑制剂库，以探测每种蛋白激酶抑制剂作为依托泊苷诱导DNA损伤的增强剂。在这里，我们发现3-甲基腺嘌呤显著增加依托泊苷诱导的DNA损伤水平。我们的研究结果表明，384孔的CometChip可用于大规模DNA损伤分析，这可能在评估化疗疗效、化合物文库筛选、基于人群的基因组损伤分析和评估环境基因毒素对基因组完整性的影响方面具有更大的潜力。
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.
影响因子：
2.8
作者：
Sykora P;Chiari Y;Heaton A;Moreno N;Glaberman S;Sobol RW
通讯作者：
Sobol RW