Development of a procedure for high content and higher throughput analysis of DNA-damage based on the COMET-assay

The sensitivity, specificity and relevance of the COMET-assay as a method for determination of DNA damage in vivo and in vitro have been highlighted in many studies. Methodical variations have been introduced addressing specific points of investigations such as the nature of strand breaks or the detection of DNA base modifications. Actually, efforts are made to include this method in a panel of tests on genotoxicity for regulatory purposes. However, the standard COMET-assay is a time-consuming procedure leading to reduced data due to standard gel/slide preparation and analysis/quantification methods. This limitation is addressed by the development presented here. A method for the preparation of COMET-slides each including 20 spots of single COMET gels and high content analysis using the Olympus ScanR screening station is developed. The procedure includes (1) a simplified procedure for slide-production, (2) a smaller amount of cells needed for analysis, (3) a higher amount of COMETs quantified, (4) a fully automated analysis of COMETs including improved reanalysis, storing, visualisation and documentation possibilities, and (5) standard Comet quantification models such as the measurement of tail length or tail moment. Individual modifications of the standard COMET procedure (sample preparation, COMET quantification) can be implemented. The procedure will be extended to the analysis of up to 96 spots per slide and adapted to roboter-handling in future. Results prove the reproducibility and flexibility of the procedure and characterize it as a way to include the COMET-Assay as a fully automated analysis method in higher throughput applications and to improve standardisation possibilities.