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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

DNA damage in reference standards, assayed in a series of experiments to measure DNA damage in PBMN cells from a human biomonitoring study. Aliquots of human lymphocytes from a single batch, either untreated (red squares), or treated with Ro 19-8022 to induce 8-oxoGua (blue circles), were included as standards in each of the experiments alongside the test samples (the results of which are not shown) and analyzed for strand breaks and Fpg-sensitive sites, respectively.
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Figure 2: DNA damage in reference standards, assayed in a series of experiments to measure DNA damage in PBMN cells from a human biomonitoring study. Aliquots of human lymphocytes from a single batch, either untreated (red squares), or treated with Ro 19-8022 to induce 8-oxoGua (blue circles), were included as standards in each of the experiments alongside the test samples (the results of which are not shown) and analyzed for strand breaks and Fpg-sensitive sites, respectively.

Mentions: Standards are particularly important when many samples (for example, from a human biomonitoring trial) are analyzed in a series of experiments over an extended period. Figure 2 shows typical results from such a trial; standards—either untreated, or treated with Ro 19-8022 plus light to induce 8-oxoGua—were included in each experiment. The CV for the untreated cells was 52%, while for the treated cells it was 14%. Variation in experiments carried out over a long time period can be expected to be greater than variation within an experiment. Figure 2 also illustrates the point made above, that where levels of damage are close to the limit of detection (0% tail DNA), the relative variation will be greater.


Controlling variation in the comet assay.

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

DNA damage in reference standards, assayed in a series of experiments to measure DNA damage in PBMN cells from a human biomonitoring study. Aliquots of human lymphocytes from a single batch, either untreated (red squares), or treated with Ro 19-8022 to induce 8-oxoGua (blue circles), were included as standards in each of the experiments alongside the test samples (the results of which are not shown) and analyzed for strand breaks and Fpg-sensitive sites, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: DNA damage in reference standards, assayed in a series of experiments to measure DNA damage in PBMN cells from a human biomonitoring study. Aliquots of human lymphocytes from a single batch, either untreated (red squares), or treated with Ro 19-8022 to induce 8-oxoGua (blue circles), were included as standards in each of the experiments alongside the test samples (the results of which are not shown) and analyzed for strand breaks and Fpg-sensitive sites, respectively.
Mentions: Standards are particularly important when many samples (for example, from a human biomonitoring trial) are analyzed in a series of experiments over an extended period. Figure 2 shows typical results from such a trial; standards—either untreated, or treated with Ro 19-8022 plus light to induce 8-oxoGua—were included in each experiment. The CV for the untreated cells was 52%, while for the treated cells it was 14%. Variation in experiments carried out over a long time period can be expected to be greater than variation within an experiment. Figure 2 also illustrates the point made above, that where levels of damage are close to the limit of detection (0% tail DNA), the relative variation will be greater.

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