Ulster University Logo

Ulster Institutional Repository

Systematic random sampling of the comet assay

Biomedical Sciences Research Institute Computer Science Research Institute Environmental Sciences Research Institute Nanotechnology & Advanced Materials Research Institute

McArt, Darragh G, Wasson, Gillian R, McKerr, George, Saetzler, Kurt, Reed, Matt and Howard, Vyvyan (2009) Systematic random sampling of the comet assay. Mutagenesis, 24 (4). pp. 373-378. [Journal article]

WarningThere is a more recent version of this item available.

Full text not available from this repository.

URL: http://mutage.oxfordjournals.org/cgi/content/abstract/24/4/373

DOI: 10.1093/mutage/gep020

Abstract

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.

Item Type:Journal article
Faculties and Schools:Faculty of Life and Health Sciences
Faculty of Life and Health Sciences > School of Biomedical Sciences
Research Institutes and Groups:Biomedical Sciences Research Institute
Biomedical Sciences Research Institute > Molecular Medicine
Biomedical Sciences Research Institute > Molecular Medicine > Nano Systems Biology
Biomedical Sciences Research Institute > Molecular Medicine > Neuroscience & Neurodegenerative Diseases
ID Code:3773
Deposited By:Dr Kurt Saetzler
Deposited On:01 Feb 2010 10:19
Last Modified:15 May 2012 13:00

Available Versions of this Item

Repository Staff Only: item control page