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

Mahsa Karbaschi,Yunhee Ji,Helen G Tempest,Alhanoof Alohaly,Abdulhadi Mohammed S Abdulwahed,Shanna L Burke,Marcus S Cooke,Thomas M Boulos,Juan F Bedoya

doi:10.3390/ijms20236072

Mahsa Karbaschi, Yunhee Ji + Show 7 more

Open Access

https://doi.org/10.3390/ijms20236072

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Abstract

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.

Highlights

Of all the methodologies for genotoxicity testing, single cell gel electrophoresis has become one of the most popular DNA damage assessment tools by the virtue of its sensitivity, adaptability, economy, ease of use, reproducibility, and reliability [1]
We evaluated factors associated with key steps in the alkaline comet assay process: Lysis buffer shelf life, neutralization buffer shelf life, lysis buffer temperature, incubation duration, and airdrying of slides
After 30 min, the media containing H2O2 were removed from the wells and the cells washed with PBS twice to remove any possible residual H2O2, and the cells harvested for processing in the comet assay

Summary

Introduction

Of all the methodologies for genotoxicity testing, single cell gel electrophoresis (the comet assay) has become one of the most popular DNA damage assessment tools by the virtue of its sensitivity, adaptability, economy, ease of use, reproducibility, and reliability [1]. The comet assay detects strand breaks and alkali-labile sites (which become strand breaks under alkali conditions) [2], arising from the interaction of various damaging intermediates (e.g., reactive oxygen species, e.g., hydroxyl radicals and hydrogen peroxide) with DNA [3], following exposure to a wide variety of genotoxins, such as ultraviolet radiation. This has led to its widespread application to in vitro and in vivo genotoxicity testing, e.g., nanomaterials [4], together with human biomonitoring [5], in environmental.

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