Measuring DNA modifications with the comet assay: a compendium of protocols

A. Collins; P. Moller; G. Gajski; S. Vodenkova; A. Abdulwahed; D. Anderson; E.E. Bankoglu; S. Bonassi; E. Boutet-Robinet; G. Brunborg; C. Chao; M.S. Cooke; C. Costa; S. Costa; A. Dhawan; J. de Lapuente; C. Del Bo; J. Dubus; M. Dusinska; S.J. Duthie; N. El Yamani; B. Engelward; I. Gaivao; L. Giovannelli; R. Godschalk; S. Guilherme; K.B. Gutzkow; K. Habas; A. Hernandez; O. Herrero; M. Isidori; A.N. Jha; S. Knasmuller; I.M. Kooter; G. Koppen; M. Kruszewski; C. Ladeira; B. Laffon; M. Larramendy; L. Le Hegarat; A. Lewies; A. Lewinska; G.E. Liwszyc; A.L. de Cerain; M. Manjanatha; R. Marcos; M. Milic; V.M. de Andrade; M. Moretti; D. Muruzabal; Amaya Azqueta

doi:10.1038/s41596-022-00754-y

Measuring DNA modifications with the comet assay: a compendium of protocols

A. Collins, P. Moller, G. Gajski, S. Vodenkova, A. Abdulwahed, D. Anderson, E.E. Bankoglu, S. Bonassi, E. Boutet-Robinet, G. Brunborg, C. Chao, M.S. Cooke, C. Costa, S. Costa, A. Dhawan, J. de Lapuente, C. Del Bo, J. Dubus, M. Dusinska, S.J. DuthieN. El Yamani, B. Engelward, I. Gaivao, L. Giovannelli, R. Godschalk, S. Guilherme, K.B. Gutzkow, K. Habas, A. Hernandez, O. Herrero, M. Isidori, A.N. Jha, S. Knasmuller, I.M. Kooter, G. Koppen, M. Kruszewski, C. Ladeira, B. Laffon, M. Larramendy, L. Le Hegarat, A. Lewies, A. Lewinska, G.E. Liwszyc, A.L. de Cerain, M. Manjanatha, R. Marcos, M. Milic, V.M. de Andrade, M. Moretti, D. Muruzabal, Amaya Azqueta^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Original language	English
Pages (from-to)	929-989
Number of pages	61
Journal	Nature Protocols
Volume	18
Issue number	3
Early online date	1 Jan 2023
DOIs	https://doi.org/10.1038/s41596-022-00754-y
Publication status	Published - Mar 2023

Keywords

CELL GEL-ELECTROPHORESIS
DOUBLE-STRAND BREAKS
IN-VIVO MODEL
INTER-LABORATORY VARIATION
INDUCED OXIDATIVE STRESS
INTERSTRAND CROSS-LINKS
ENABLES HIGH-THROUGHPUT
DROSOPHILA-MELANOGASTER
EPITHELIAL-CELLS
CORD BLOOD

Access to Document

10.1038/s41596-022-00754-y

Cite this

Collins, A., Moller, P., Gajski, G., Vodenkova, S., Abdulwahed, A., Anderson, D., Bankoglu, E. E., Bonassi, S., Boutet-Robinet, E., Brunborg, G., Chao, C., Cooke, M. S., Costa, C., Costa, S., Dhawan, A., de Lapuente, J., Del Bo, C., Dubus, J., Dusinska, M., ... Azqueta, A. (2023). Measuring DNA modifications with the comet assay: a compendium of protocols. Nature Protocols, 18(3), 929-989. https://doi.org/10.1038/s41596-022-00754-y

@article{8a88866e0599454da2e4cfe8b76a1c69,

title = "Measuring DNA modifications with the comet assay: a compendium of protocols",

abstract = "The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.",

keywords = "CELL GEL-ELECTROPHORESIS, DOUBLE-STRAND BREAKS, IN-VIVO MODEL, INTER-LABORATORY VARIATION, INDUCED OXIDATIVE STRESS, INTERSTRAND CROSS-LINKS, ENABLES HIGH-THROUGHPUT, DROSOPHILA-MELANOGASTER, EPITHELIAL-CELLS, CORD BLOOD",

author = "A. Collins and P. Moller and G. Gajski and S. Vodenkova and A. Abdulwahed and D. Anderson and E.E. Bankoglu and S. Bonassi and E. Boutet-Robinet and G. Brunborg and C. Chao and M.S. Cooke and C. Costa and S. Costa and A. Dhawan and {de Lapuente}, J. and {Del Bo}, C. and J. Dubus and M. Dusinska and S.J. Duthie and {El Yamani}, N. and B. Engelward and I. Gaivao and L. Giovannelli and R. Godschalk and S. Guilherme and K.B. Gutzkow and K. Habas and A. Hernandez and O. Herrero and M. Isidori and A.N. Jha and S. Knasmuller and I.M. Kooter and G. Koppen and M. Kruszewski and C. Ladeira and B. Laffon and M. Larramendy and {Le Hegarat}, L. and A. Lewies and A. Lewinska and G.E. Liwszyc and {de Cerain}, A.L. and M. Manjanatha and R. Marcos and M. Milic and {de Andrade}, V.M. and M. Moretti and D. Muruzabal and Amaya Azqueta",

note = "Funding Information: We thank the hCOMET project (COST Action, CA 15132) for support. A. Azqueta thanks the Ministry of Science and Innovation (AGL2015-70640-R and PID2020-115348RB-I00) of the Spanish Government. S.G. thanks the national funds (OE), through FCT—Funda{\c c}{\~a}o para a Ci{\^e}ncia e a Tecnologia (IP, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July) for personal support. V.M.d.A. thanks the National Council of Technological and Scientific Development (CNPq—304203/2018-1) for personal support. D.M. thanks the program {\textquoteleft}Ayudas para la formaci{\'o}n de profesorado universitario (FPU){\textquoteright} of the Spanish Government for the predoctoral grant received. N.O. thanks the NIEHS Superfund Research Program ES ES027707 for personal support. J.S.-S. thanks the Government of Navarra for the predoctoral grant received. V.V. thanks the Ministerio de Educaci{\'o}n, Cultura y Deporte ({\textquoteleft}Beatriz Galindo{\textquoteright} program, BEAGAL18/00142) of the Spanish Government for personal support. M.S.C. acknowledges personal support from the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274-01. This paper reflects the views of the authors and does not necessarily reflect those of the US Food and Drug Administration or the National Institutes of Health. Funding Information: We thank the hCOMET project (COST Action, CA 15132) for support. A. Azqueta thanks the Ministry of Science and Innovation (AGL2015-70640-R and PID2020-115348RB-I00) of the Spanish Government. S.G. thanks the national funds (OE), through FCT—Funda{\c c}{\~a}o para a Ci{\^e}ncia e a Tecnologia (IP, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July) for personal support. V.M.d.A. thanks the National Council of Technological and Scientific Development (CNPq—304203/2018-1) for personal support. D.M. thanks the program {\textquoteleft}Ayudas para la formaci{\'o}n de profesorado universitario (FPU){\textquoteright} of the Spanish Government for the predoctoral grant received. N.O. thanks the NIEHS Superfund Research Program ES ES027707 for personal support. J.S.-S. thanks the Government of Navarra for the predoctoral grant received. V.V. thanks the Ministerio de Educaci{\'o}n, Cultura y Deporte ({\textquoteleft}Beatriz Galindo{\textquoteright} program, BEAGAL18/00142) of the Spanish Government for personal support. M.S.C. acknowledges personal support from the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274-01. This paper reflects the views of the authors and does not necessarily reflect those of the US Food and Drug Administration or the National Institutes of Health. Publisher Copyright: {\textcopyright} 2023, Springer Nature Limited.",

year = "2023",

month = mar,

doi = "10.1038/s41596-022-00754-y",

language = "English",

volume = "18",

pages = "929--989",

journal = "Nature Protocols",

issn = "1754-2189",

publisher = "Nature Publishing Group",

number = "3",

}

Collins, A, Moller, P, Gajski, G, Vodenkova, S, Abdulwahed, A, Anderson, D, Bankoglu, EE, Bonassi, S, Boutet-Robinet, E, Brunborg, G, Chao, C, Cooke, MS, Costa, C, Costa, S, Dhawan, A, de Lapuente, J, Del Bo, C, Dubus, J, Dusinska, M, Duthie, SJ, El Yamani, N, Engelward, B, Gaivao, I, Giovannelli, L, Godschalk, R, Guilherme, S, Gutzkow, KB, Habas, K, Hernandez, A, Herrero, O, Isidori, M, Jha, AN, Knasmuller, S, Kooter, IM, Koppen, G, Kruszewski, M, Ladeira, C, Laffon, B, Larramendy, M, Le Hegarat, L, Lewies, A, Lewinska, A, Liwszyc, GE, de Cerain, AL, Manjanatha, M, Marcos, R, Milic, M, de Andrade, VM, Moretti, M, Muruzabal, D & Azqueta, A 2023, 'Measuring DNA modifications with the comet assay: a compendium of protocols', Nature Protocols, vol. 18, no. 3, pp. 929-989. https://doi.org/10.1038/s41596-022-00754-y

TY - JOUR

T1 - Measuring DNA modifications with the comet assay: a compendium of protocols

AU - Collins, A.

AU - Moller, P.

AU - Gajski, G.

AU - Vodenkova, S.

AU - Abdulwahed, A.

AU - Anderson, D.

AU - Bankoglu, E.E.

AU - Bonassi, S.

AU - Boutet-Robinet, E.

AU - Brunborg, G.

AU - Chao, C.

AU - Cooke, M.S.

AU - Costa, C.

AU - Costa, S.

AU - Dhawan, A.

AU - de Lapuente, J.

AU - Del Bo, C.

AU - Dubus, J.

AU - Dusinska, M.

AU - Duthie, S.J.

AU - El Yamani, N.

AU - Engelward, B.

AU - Gaivao, I.

AU - Giovannelli, L.

AU - Godschalk, R.

AU - Guilherme, S.

AU - Gutzkow, K.B.

AU - Habas, K.

AU - Hernandez, A.

AU - Herrero, O.

AU - Isidori, M.

AU - Jha, A.N.

AU - Knasmuller, S.

AU - Kooter, I.M.

AU - Koppen, G.

AU - Kruszewski, M.

AU - Ladeira, C.

AU - Laffon, B.

AU - Larramendy, M.

AU - Le Hegarat, L.

AU - Lewies, A.

AU - Lewinska, A.

AU - Liwszyc, G.E.

AU - de Cerain, A.L.

AU - Manjanatha, M.

AU - Marcos, R.

AU - Milic, M.

AU - de Andrade, V.M.

AU - Moretti, M.

AU - Muruzabal, D.

AU - Azqueta, Amaya

N1 - Funding Information: We thank the hCOMET project (COST Action, CA 15132) for support. A. Azqueta thanks the Ministry of Science and Innovation (AGL2015-70640-R and PID2020-115348RB-I00) of the Spanish Government. S.G. thanks the national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia (IP, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July) for personal support. V.M.d.A. thanks the National Council of Technological and Scientific Development (CNPq—304203/2018-1) for personal support. D.M. thanks the program ‘Ayudas para la formación de profesorado universitario (FPU)’ of the Spanish Government for the predoctoral grant received. N.O. thanks the NIEHS Superfund Research Program ES ES027707 for personal support. J.S.-S. thanks the Government of Navarra for the predoctoral grant received. V.V. thanks the Ministerio de Educación, Cultura y Deporte (‘Beatriz Galindo’ program, BEAGAL18/00142) of the Spanish Government for personal support. M.S.C. acknowledges personal support from the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274-01. This paper reflects the views of the authors and does not necessarily reflect those of the US Food and Drug Administration or the National Institutes of Health. Funding Information: We thank the hCOMET project (COST Action, CA 15132) for support. A. Azqueta thanks the Ministry of Science and Innovation (AGL2015-70640-R and PID2020-115348RB-I00) of the Spanish Government. S.G. thanks the national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia (IP, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July) for personal support. V.M.d.A. thanks the National Council of Technological and Scientific Development (CNPq—304203/2018-1) for personal support. D.M. thanks the program ‘Ayudas para la formación de profesorado universitario (FPU)’ of the Spanish Government for the predoctoral grant received. N.O. thanks the NIEHS Superfund Research Program ES ES027707 for personal support. J.S.-S. thanks the Government of Navarra for the predoctoral grant received. V.V. thanks the Ministerio de Educación, Cultura y Deporte (‘Beatriz Galindo’ program, BEAGAL18/00142) of the Spanish Government for personal support. M.S.C. acknowledges personal support from the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274-01. This paper reflects the views of the authors and does not necessarily reflect those of the US Food and Drug Administration or the National Institutes of Health. Publisher Copyright: © 2023, Springer Nature Limited.

PY - 2023/3

Y1 - 2023/3

N2 - The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

AB - The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

KW - CELL GEL-ELECTROPHORESIS

KW - DOUBLE-STRAND BREAKS

KW - IN-VIVO MODEL

KW - INTER-LABORATORY VARIATION

KW - INDUCED OXIDATIVE STRESS

KW - INTERSTRAND CROSS-LINKS

KW - ENABLES HIGH-THROUGHPUT

KW - DROSOPHILA-MELANOGASTER

KW - EPITHELIAL-CELLS

KW - CORD BLOOD

U2 - 10.1038/s41596-022-00754-y

DO - 10.1038/s41596-022-00754-y

M3 - Article

C2 - 36707722

SN - 1754-2189

VL - 18

SP - 929

EP - 989

JO - Nature Protocols

JF - Nature Protocols

IS - 3

ER -