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Open AccessArticle

Evaluation of the Major Steps in the Conventional Protocol for the Alkaline Comet Assay

1
Oxidative Stress Group, Department of Environmental Health Sciences, Florida International University, Miami, FL 33199, USA
2
Department of Human and Molecular Genetics, Florida International University, Miami, FL 33199, USA
3
Department of Dietetics and Nutrition, Florida International University, Miami, FL 33199, USA
4
School of Social Work, Florida International University, Miami, FL 33199, USA
5
Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(23), 6072; https://doi.org/10.3390/ijms20236072
Received: 23 October 2019 / Revised: 26 November 2019 / Accepted: 28 November 2019 / Published: 2 December 2019
(This article belongs to the Collection Feature Papers in Molecular Toxicology)
Single cell gel electrophoresis, also known as the comet assay, has become a widespread DNA damage assessment tool due to its sensitivity, adaptability, low cost, ease of use, and reliability. Despite these benefits, this assay has shortcomings, such as long assay running time, the manipulation of multiple slides, individually, through numerous process steps, the challenge of working in a darkened environment, and reportedly considerable inter- and intra-laboratory variation. All researchers typically perform the comet assay based upon a common core approach; however, it appears that some steps in this core have little proven basis, and may exist, partly, out of convenience, or dogma. The aim of this study was to critically re-evaluate key steps in the comet assay, using our laboratory’s protocol as a model, firstly to understand the scientific basis for why certain steps in the protocol are performed in a particular manner, and secondly to simplify the assay, and decrease the cost and run time. Here, the shelf life of the lysis and neutralization buffers, the effect of temperature and incubation period during the lysis step, the necessity for drying the slides between the electrophoresis and staining step, and the need to perform the sample workup and electrophoresis steps under subdued light were all evaluated. View Full-Text
Keywords: comet assay; DNA damage; DNA repair; oxidative stress; genotoxicity; human biomonitoring comet assay; DNA damage; DNA repair; oxidative stress; genotoxicity; human biomonitoring
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MDPI and ACS Style

Karbaschi, M.; Ji, Y.; Abdulwahed, A.M.S.; Alohaly, A.; Bedoya, J.F.; Burke, S.L.; Boulos, T.M.; Tempest, H.G.; Cooke, M.S. Evaluation of the Major Steps in the Conventional Protocol for the Alkaline Comet Assay. Int. J. Mol. Sci. 2019, 20, 6072.

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