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Keywords
(7)
Comet Assay
Dna Damage
Human Lymphocyte
Image Analysis
Regression Analysis
Arbitrary Units
Compact Disc
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Visual estimation of the percentage of DNA in the tail in the comet assay: Evaluation of different approaches in an intercomparison exercise
Visual estimation of the percentage of DNA in the tail in the comet assay: Evaluation of different approaches in an intercomparison exercise,10.1016/j
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Visual estimation of the percentage of DNA in the tail in the comet assay: Evaluation of different approaches in an intercomparison exercise
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Omar García
,
Ivonne Romero
,
Jorge Ernesto González
,
Damaris L. Moreno
,
Elizabeth Cuétara
,
Yesenia Rivero
,
Ariadne Gutiérrez
,
Carlos L. Pérez
,
Aimée Álvarez
,
Deyanira Carnesolta
,
Irania Guevara
One of the difficulties in the comparison of results between laboratories working with the
comet assay
is the great diversity of parameters used to express
DNA damage
and the lack of conversion factors between the majority of them. Here we report a scorer-independent conversion curve to transform the values of
DNA damage
reported in
arbitrary units
(AU) into estimated percentage of DNA in the tail (E%T), and the results obtained in an intercomparison exercise where the effectiveness of this curve and two others proposed in the literature (E%T=AU/4 and E%T=(AU/5)+10) were tested. To obtain the conversion curve, human lymphocytes were first treated with radiation or H2O2. Percentage of DNA in tail (%T) was then measured in 2100 comets (300comets per treatment) using Casp
image analysis
software. Subsequently, using these values of %T, categories of 0, 1, 2, 3, and 4 were assigned to comets with %T [0–1), [1–25), [25–45), [45–70), and >70, and
DNA damage
was calculated in AU, as usual.
DNA damage
was induced in the interval 24–315AU (1.54–65.23%T). The best-fit conversion curve obtained by
regression analysis
was E%T=(AU−25.87)/4.46. In the intercomparison exercise, ten scorers from nine laboratories analyzed the same comet images (recorded on a compact disc) visually. The values reported in comet categories were transformed into AU and subsequently into E%T, using the three approaches mentioned above. The best agreement between E%T and %T measured by the software (S%T) was obtained with the conversion curve reported here, where the slope of E%T versus S%T from the ten scorers was not different from 1. Using this conversion curve, the overall mean difference between E%T and S%T was 1.4±2.62 and 57 (81%) of E%T values differ from S%T by less than 5units. These findings show the strength of the scorer-independent conversion curve as a tool to compare results reported in AU or %T by different laboratories.
Journal:
Mutation Research-genetic Toxicology and Environmental Mutagenesis - MUTAT RES-GENET TOXICOL E M
, vol. 720, no. 1, pp. 14-21, 2011
DOI:
10.1016/j.mrgentox.2010.11.011
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