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Mutagenesis Advance Access originally published online on May 28, 2009
Mutagenesis 2009 24(4):373-378; doi:10.1093/mutage/gep020
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© The Author 2009. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Systematic random sampling of the comet assay

Darragh G. McArt, Gillian R. Wasson, George McKerr, Kurt Saetzler, Matt Reed and C. Vyvyan Howard*

Biomedical Sciences Research Institute, University of Ulster, Coleraine, County Londonderry BT52 1SA, Northern Ireland, UK

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory ‘tail’ DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the ‘randomness’ of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

* To whom correspondence should be addressed. Tel: +44 287 032 3383; Fax: +44 287 0322 4965; Email: v.howard{at}ulster.ac.uk

Received on January 6, 2009; revised on April 28, 2009; accepted on April 28, 2009.


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