Nucleic Acids Research Advance Access published online on October 14, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp826
Methods Online |
Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair
1Radiation and Oxidative Stress Group, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK, 2Herbal Medicine Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia, 3IPATIMUP, Rua Dr. Roberto Frias, Porto, Portugal, 4Alexander Rapp, Molecular Cell Biology Group, Department of Biology, Technische Universitaet Darmstadt, Schnittspahnstrasse 10, D-64287 Darmstadt, Germany
*To whom correspondence should be addressed. Tel: +44 116 223 1841; Fax: +44 116 223 1840; Email: gdj2{at}le.ac.uk
Received April 24, 2009. Revised July 16, 2009. Accepted September 19, 2009.
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.
The members of the ECVAG are given in the Acknowledgements. Present addresses: M. Zainol, Herbal Medicine Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia.
Gabriela M. Almeida, IPATIMUP, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal.