Automated analysis of DNA damage in the high-throughput version of the comet assay

Recently a high-throughput version of the comet assay was developed using a special 96-well multichamber plate (MCP) [1]. In this version, the electrophoresis is performed directly on the MCP, which makes transferring of cells to microscope slides unnecessary. In order to facilitate the scoring proc...

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Bibliographic Details
Published in:Mutation research. Genetic toxicology and environmental mutagenesis 2010-04, Vol.698 (1), p.1-5
Main Authors: Stang, A., Brend́amour, M., Schunck, C., Witte, I.
Format: Article
Language:English
Subjects:
Automated analysis
Biological and medical sciences
Cells
Comet assay
Comparative analysis
Deoxyribonucleic acid
DNA
DNA damage
Fundamental and applied biological sciences. Psychology
Genetics of eukaryotes. Biological and molecular evolution
High-throughput
Medical sciences
Olea
Studies
Toxicology
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Summary:Recently a high-throughput version of the comet assay was developed using a special 96-well multichamber plate (MCP) [1]. In this version, the electrophoresis is performed directly on the MCP, which makes transferring of cells to microscope slides unnecessary. In order to facilitate the scoring procedure we adapted an automated slide-scanning system (Metafer MetaCyte with CometScan) to enable unattended analysis of comets on the MCP. The results of the system were compared with the data obtained with two interactive comet-assay analysis systems. For induction of DNA damage in human fibroblasts methylmethane sulfonate (MMS) or H 2O 2 was used. The three systems revealed similar, concentration-dependent results for all parameters tested: tail moment (tm), % DNA-in-tail and olive tail moment. Near the detection limit of 5–6% DNA-in-tail a significant difference with the untreated control was obtained by use of four parallel samples ( p = 0.01). With the newly developed automated analysis system, the evaluation of either 50 or 100 comets yielded similar standard errors for either treatment with MMS or H 2O 2, thus showing that the method is suitable to reveal the crucial low-dose effects with high precision. The results also show that the time needed for automated evaluation of comets on the MCP was reduced by a factor of 10 when compared with the time required for interactive evaluation. In summary, the high-throughput version of the comet assay combined with the automated evaluating system increased the output by a factor up to 180 compared with the standard method.
ISSN:1383-5718
1879-3592
DOI:10.1016/j.mrgentox.2010.02.014