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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

Normalization of comet assay data. Results of analysis of 30 lymphocyte samples using Fpg to detect 8-oxoGua were corrected for variation as indicated by reference standards (see text). Data are shown before (green squares) and after (purple circles) normalization. Results for samples 5 and 9 changed substantially after normalization.
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Figure 3: Normalization of comet assay data. Results of analysis of 30 lymphocyte samples using Fpg to detect 8-oxoGua were corrected for variation as indicated by reference standards (see text). Data are shown before (green squares) and after (purple circles) normalization. Results for samples 5 and 9 changed substantially after normalization.

Mentions: Figure 3 gives an example of normalization. Samples of lymphocytes from an intervention study were analyzed for Fpg-sensitive sites. The data were then corrected for variation as indicated by positive reference standards run in the same experiments. In most cases, normalization made little difference, but substantial changes were seen in a few samples, namely 5 and 9 in this set of samples.


Controlling variation in the comet assay.

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

Normalization of comet assay data. Results of analysis of 30 lymphocyte samples using Fpg to detect 8-oxoGua were corrected for variation as indicated by reference standards (see text). Data are shown before (green squares) and after (purple circles) normalization. Results for samples 5 and 9 changed substantially after normalization.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Normalization of comet assay data. Results of analysis of 30 lymphocyte samples using Fpg to detect 8-oxoGua were corrected for variation as indicated by reference standards (see text). Data are shown before (green squares) and after (purple circles) normalization. Results for samples 5 and 9 changed substantially after normalization.
Mentions: Figure 3 gives an example of normalization. Samples of lymphocytes from an intervention study were analyzed for Fpg-sensitive sites. The data were then corrected for variation as indicated by positive reference standards run in the same experiments. In most cases, normalization made little difference, but substantial changes were seen in a few samples, namely 5 and 9 in this set of samples.

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