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Controlling variation in the comet assay.

Collins AR, El Yamani N, Lorenzo Y, Shaposhnikov S, Brunborg G, Azqueta A - Front Genet (2014)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition, Faculty of Medicine, University of Oslo Oslo, Norway.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of different voltage gradients at constant current (A) and different electrophoresis times (B) on the % tail DNA in TK-6 cells, untreated (dark shading), and treated with 70 μM H2O2 (light shading). The mean and range of values from two experiments are shown. Redrawn from Azqueta et al. (2011a) with permission from Elsevier.
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Figure 1: Effect of different voltage gradients at constant current (A) and different electrophoresis times (B) on the % tail DNA in TK-6 cells, untreated (dark shading), and treated with 70 μM H2O2 (light shading). The mean and range of values from two experiments are shown. Redrawn from Azqueta et al. (2011a) with permission from Elsevier.

Mentions: For comets from irradiated or H2O2-treated cells, the % tail DNA is strongly influenced by both electrophoresis time (varied up to 40 min) and voltage gradient (between <0.2 and 1.6 V/cm) (Ersson and Möller, 2011; Azqueta et al., 2011a) (Figure 1). To a certain extent, a low voltage gradient for a long time will give similar results to a higher voltage gradient for a shorter time. The voltage gradient should be measured over the platform on which the slides are placed rather than between the electrodes, since that is where the electric potential pulls out damaged DNA from the nucleoids. Between the electrode and platform edge, in standard tanks, there is a relatively deep trough of electrophoresis solution, with low resistance, so that the voltage drop is much lower than over the platform where there is a shallow layer of solution over the slides; hence, measuring the total applied voltage and dividing it by the distance between the electrodes gives an erroneous V/cm value. Increasing the depth of solution over the platform increases the current (because the resistance is decreased), and this causes a slight decrease in the % tail DNA—an effect explained by the reduced voltage drop over the platform (Azqueta et al., 2011a). Current itself does not influence DNA migration. These comments relate to tanks with electrodes in troughs and a central platform. There are few restrictions in tank design as long as the voltage gradient is constant where the samples are placed for electrophoresis. We recommend careful measurement of the voltage gradient at the relevant position and depth, particularly when non-standard electrophoresis tanks are being used.


Controlling variation in the comet assay.

Collins AR, El Yamani N, Lorenzo Y, Shaposhnikov S, Brunborg G, Azqueta A - Front Genet (2014)

Effect of different voltage gradients at constant current (A) and different electrophoresis times (B) on the % tail DNA in TK-6 cells, untreated (dark shading), and treated with 70 μM H2O2 (light shading). The mean and range of values from two experiments are shown. Redrawn from Azqueta et al. (2011a) with permission from Elsevier.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202776&req=5

Figure 1: Effect of different voltage gradients at constant current (A) and different electrophoresis times (B) on the % tail DNA in TK-6 cells, untreated (dark shading), and treated with 70 μM H2O2 (light shading). The mean and range of values from two experiments are shown. Redrawn from Azqueta et al. (2011a) with permission from Elsevier.
Mentions: For comets from irradiated or H2O2-treated cells, the % tail DNA is strongly influenced by both electrophoresis time (varied up to 40 min) and voltage gradient (between <0.2 and 1.6 V/cm) (Ersson and Möller, 2011; Azqueta et al., 2011a) (Figure 1). To a certain extent, a low voltage gradient for a long time will give similar results to a higher voltage gradient for a shorter time. The voltage gradient should be measured over the platform on which the slides are placed rather than between the electrodes, since that is where the electric potential pulls out damaged DNA from the nucleoids. Between the electrode and platform edge, in standard tanks, there is a relatively deep trough of electrophoresis solution, with low resistance, so that the voltage drop is much lower than over the platform where there is a shallow layer of solution over the slides; hence, measuring the total applied voltage and dividing it by the distance between the electrodes gives an erroneous V/cm value. Increasing the depth of solution over the platform increases the current (because the resistance is decreased), and this causes a slight decrease in the % tail DNA—an effect explained by the reduced voltage drop over the platform (Azqueta et al., 2011a). Current itself does not influence DNA migration. These comments relate to tanks with electrodes in troughs and a central platform. There are few restrictions in tank design as long as the voltage gradient is constant where the samples are placed for electrophoresis. We recommend careful measurement of the voltage gradient at the relevant position and depth, particularly when non-standard electrophoresis tanks are being used.

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition, Faculty of Medicine, University of Oslo Oslo, Norway.

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.

No MeSH data available.


Related in: MedlinePlus