Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial
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Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial. / Møller, Peter; Azqueta, Amaya; Collia, Miguel; Bakuradze, Tamara; Richling, Elke; Bankoglu, Ezgi Eyluel; Stopper, Helga; Bastos, Victoria Claudino; Langie, Sabine A S; Jensen, Annie; Ristori, Sara; Scavone, Francesca; Giovannelli, Lisa; Wojewódzka, Maria; Kruszewski, Marcin; Valdiglesias, Vanessa; Laffon, Blanca; Costa, Carla; Costa, Solange; Teixeira, João Paulo; Marino, Mirko; Del Bo', Cristian; Riso, Patrizia; Zhang, Congying; Shaposhnikov, Sergey; Collins, Andrew.
In: Mutagenesis, Vol. 38, No. 5, 2023, p. 283–294.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial
AU - Møller, Peter
AU - Azqueta, Amaya
AU - Collia, Miguel
AU - Bakuradze, Tamara
AU - Richling, Elke
AU - Bankoglu, Ezgi Eyluel
AU - Stopper, Helga
AU - Bastos, Victoria Claudino
AU - Langie, Sabine A S
AU - Jensen, Annie
AU - Ristori, Sara
AU - Scavone, Francesca
AU - Giovannelli, Lisa
AU - Wojewódzka, Maria
AU - Kruszewski, Marcin
AU - Valdiglesias, Vanessa
AU - Laffon, Blanca
AU - Costa, Carla
AU - Costa, Solange
AU - Teixeira, João Paulo
AU - Marino, Mirko
AU - Del Bo', Cristian
AU - Riso, Patrizia
AU - Zhang, Congying
AU - Shaposhnikov, Sergey
AU - Collins, Andrew
N1 - © The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
PY - 2023
Y1 - 2023
N2 - The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) indicates importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.
AB - The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) indicates importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.
U2 - 10.1093/mutage/gead014
DO - 10.1093/mutage/gead014
M3 - Journal article
C2 - 37228081
VL - 38
SP - 283
EP - 294
JO - Mutagenesis
JF - Mutagenesis
SN - 0267-8357
IS - 5
ER -
ID: 359540846