University of Leicester
Nucl. Acids Res.-2009-Zainol-e150.pdf (3.32 MB)
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Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair.

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posted on 2012-10-24, 09:00 authored by Julia Stoute, Gabriela M. Almeida, A. Rapp, Karen J. Bowman, George D. D. Jones, ECVAG
The Comet assay (CA) is a sensitive/simple measure of genotoxicity. However, many features of CA contribute variability. To minimize these, we have introduced internal standard materials consisting of 'reference' cells which have their DNA substituted with BrdU. Using a fluorescent anti-BrdU antibody, plus an additional barrier filter, comets derived from these cells could be readily distinguished from the 'test'-cell comets, present in the same gel. In experiments to evaluate the reference cell comets as external and internal standards, the reference and test cells were present in separate gels on the same slide or mixed together in the same gel, respectively, before their co-exposure to X-irradiation. Using the reference cell comets as internal standards led to substantial reductions in the coefficient of variation (CoV) for intra- and inter-experimental measures of comet formation and DNA damage repair; only minor reductions in CoV were noted when the reference and test cell comets were in separate gels. These studies indicate that differences between individual gels appreciably contribute to CA variation. Further studies using the reference cells as internal standards allowed greater significance to be obtained between groups of replicate samples. Ultimately, we anticipate that development will deliver robust quality assurance materials for CA.


This work was supported by Cancer Research UK project grant (C13560/A4661) awarded to GDDJ and also by two Type B Research Projects ‘Assessment and reduction of comet assay variation in relation to DNA damage and DNA repair phenotype’ and ‘Assessment and reduction of comet assay variation in relation to DNA damage’ from ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility) [a network of excellence operating within the European Union Sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943)] awarded to the European Comet Assay Validation Group (ECVAG). MZ is supported by the Public Service Department of Malaysia and the Institute for Medical Research, Malaysia. GMA is supported by FCT, Portugal, and the European Social Fund. Funding for open access charge: Grant funding.



Nucleic Acids Research, 2009, 37 (22), pp. e150-e150

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