An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.
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Godschalk, Roger W. L.
Langie, Sabine A. S.
Jones, George D. D.
Kwok, Rachel W. L.
Phillips, David H.
Routledge, Michael N.
Charlton, Alexander J.
Collins, Andrew R.
MetadataShow full item record
AbstractThe increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.
CitationMutagenesis 2010, 25 (2):125-132
SponsorsEnvironmental Cancer Risk, Nutrition and Individual Susceptibility, a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943); the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS); the European Union 6th Framework Program strategic targeted research project ‘COMICS’ (Contract No 037575); Swedish Research Council (Vetenskapsra°det).
- Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial.
- Authors: Forchhammer L, Johansson C, Loft S, Möller L, Godschalk RW, Langie SA, Jones GD, Kwok RW, Collins AR, Azqueta A, Phillips DH, Sozeri O, Stepnik M, Palus J, Vogel U, Wallin H, Routledge MN, Handforth C, Allione A, Matullo G, Teixeira JP, Costa S, Riso P, Porrini M, Møller P
- Issue date: 2010 Mar
- An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells.
- Authors: Ersson C, Møller P, Forchhammer L, Loft S, Azqueta A, Godschalk RW, van Schooten FJ, Jones GD, Higgins JA, Cooke MS, Mistry V, Karbaschi M, Phillips DH, Sozeri O, Routledge MN, Nelson-Smith K, Riso P, Porrini M, Matullo G, Allione A, Stepnik M, Ferlińska M, Teixeira JP, Costa S, Corcuera LA, López de Cerain A, Laffon B, Valdiglesias V, Collins AR, Möller L
- Issue date: 2013 May
- Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group.
- Authors: Møller P, Möller L, Godschalk RW, Jones GD
- Issue date: 2010 Mar
- Inter-laboratory variation in DNA damage using a standard comet assay protocol.
- Authors: Forchhammer L, Ersson C, Loft S, Möller L, Godschalk RW, van Schooten FJ, Jones GD, Higgins JA, Cooke M, Mistry V, Karbaschi M, Collins AR, Azqueta A, Phillips DH, Sozeri O, Routledge MN, Nelson-Smith K, Riso P, Porrini M, Matullo G, Allione A, Stępnik M, Komorowska M, Teixeira JP, Costa S, Corcuera LA, López de Cerain A, Laffon B, Valdiglesias V, Møller P
- Issue date: 2012 Nov
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- Authors: Kain J, Karlsson HL, Möller L
- Issue date: 2012 Jul