A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

open access articleCells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

Novel method for the high-throughput processing of slides for the comet assay

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay’s low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure

Investigation of ultraviolet radiation-induced DNA damage and repair in human skin cells

UVR, by initiating the DNA damage, can lead to mutagenesis and is regarded as the prime cause of most skin cancers. Cyclobutane pyrimidine dimers (CPD) are an important form of DNA damage induced by both UVA and UVB and removed by nucleotide excision repair. Classically, the repair of CPDs is reported to be a lengthy process. The persistence of CPDs, compared to other forms of DNA damage, is understood to be a major contributory factor to their mutagenicity. In this study, using the T4endonucleaseV-modified comet assay for CPDs, the standard slow repair of UVB-induced CPDs were confirmed, but repair of UVA-induced CPDs was more rapid in human keratinocytes and fibroblasts. A rapid initial repair of CPDs over the first 6 h post-irradiation, following either UVA or UVB treatments was noted in both cell types, but whilst this slowed significantly in the UVB-irradiated cells, it continued to be rapid in the UVA-treated cells with levels approaching baseline within 36 h. Up-regulation to the baseline level of key genes associated with nucleotide excision repair in UVA-irradiated cells, compared to down regulation of the same genes in UVB-irradiated cells was noted. There were no significant differences in cell viability between the two treatments over the first 6 h post-irradiation but, at 24 h post-irradiation, viability had decreased significantly in the UVB-irradiated cells only. These data suggest that for at least the first six hours following UVB irradiation, the majority of cells are viable and capable of repair, after that time increasing numbers of cells enter apoptosis, and cease to repair the damage. Discrimination of dead/dying cells from viable cells in any repair assay is a possible element of artefact in determining the repair kinetics of CPDs. This would contribute to the apparent slow repair of CPDs widely reported in the literature.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Novel method for the high-throughput processing of slides for the comet assay

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure

United States Patent, Patent No: US9897572

Apparatuses and associated methods for manipulating an assembly of glass slides employed in cellular assay processes are provided. Each apparatus can accommodate at least one removable rack of slides to undergo electrophoresis in a comet assay. The slides can remain in the same apparatus while being subjected to a sequence of fluid staining and washing with temperature control, advantageously shortening the amount of time required for processing the slides by keeping them in the same work station for the entire duration of the assay