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The Comet Assay: Methods for Quantitative Image Analysis and Reproducibility



Donald H. Atha, Signe Braafladt, Vytautas Reipa


The comet assay,or single cell gel electrophoresis,is a sensitive and efficient technique for analyzing DNA damage in the form of single and double strand breaks in single cells. DNA alteration,from strand breaks,can lead to cancer and other health risks. The comet assay’s increased use in a wide variety of applications emphasizes the importance of accurate quantitative output that will require establishing standard assay protocols and understanding inherent sources of error and measurement variability. Likely sources of variability include the heterogeneity of the particular cells undergoing analysis, the assay protocol used to process the cells, the microscope imaging system and the software used in the computerized analysis of these images. To determine the effect of these sources of variability on the measured percentage of damaged DNA(%DNA in tail) preparations of mammalian cells with chemically and electrochemically induced DNA oxidative damage were analyzed for the effects of varying the settings of the automated microscope(Olympus BH2)and camera(Photometrics CoolSnapHQ2),as well as the ImageJ(NIH)and CometScorePro(TriTek)image analysis software. Manual image analysis revealed measurement variations as high as 40% due to microscope focus,camera exposure time and the software image intensity threshold level. Automated image analysis reduced these variations as much as three-fold,but within a narrow range of focus and exposure settings. The magnitude of variation, observed using both of these methods was highly dependent on the overall extent of DNA damage in the particular sample. Accounting for these sources of variability with determined optimal instrument settings facilitates an accurate evaluation of the cell biological variability. Additionally,the automated microscopy allowed us to obtain the required statistical evaluation of oxidative treatments and to minimize the uncertainties in determining the extent of DNA damage in a population of cultured cells.


single cell gel electrophoresis, SCGE, comet image analysis


Atha, D. , Braafladt, S. and Reipa, V. (2016), The Comet Assay: Methods for Quantitative Image Analysis and Reproducibility, Biotechniques, [online], (Accessed May 17, 2024)


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Created September 1, 2016, Updated November 10, 2018