Controlling variation in the comet assay

Abstract

Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period—for example, analysing samples of white blood cells from a large human biomonitoring trial—to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

Impact of Weight Loss Strategies on Obesity‐Induced DNA Damage

“…The activity of the enzyme was measured before the main experiment (data not shown). The extent of DNA migration attributable to Fpg‐sensitive sites was calculated in all experiments by subtraction of the corresponding enzyme buffer values …”

Water browning mediates predatory decimation of the Arctic fairy shrimp Branchinecta paludosa

“…DNA strand breaks are among the more direct and easily detectable effects which can be assessed by means of comet assays, quantifying the degree of DNA fragmentation of single nuclei (Singh et al, 1988) by single-cell gel electrophoresis. While long acknowledged as a routine tool in human toxicology (Faust et al, 2004;Collins et al, 2014a), the comet assay has also gained interest in ecotoxicology (Jha, 2008;de Lapuente et al, 2015), where fields of application range from DNA damage analysis in immortalised cell cultures, to field-sampled mammalian blood. Chaoborus nyblaei larvae were sampled from one clear water (3.9 mg TOC L À1 ) and one brownwater pond (10.5 mg TOC L À1 ).…”

Application of the CometChip platform to assess DNA damage in field‐collected blood samples from turtles

“…Results from experiments measuring the inter laboratory variability of the classic comet assay have proven so large that any actual population differences would be potentially masked (European Standards Committee on Oxidative DNA Damage 2003; Gedik et al 2005). However, introduction of normalization parameters has improved variability from lab to lab (Collins et al 2014). An objective of this study was also to create a standardized workflow that allowed a researcher to compare samples taken over extended periods of time (weeks, months, years).…”