Technical aspects of the electrophoresis stage in the comet assay

Cover Page

Cite item

Full Text

Abstract

Inter- and intralaboratory variability of results is still a serious issue in the comet assay. There are several technical conditions of procedure, which may critically affect the results and electrophoresis terms were identified as main. A comparative assessment of the expected and actual electric field strength in five electrophoretic tanks and the contribution of the revealed differences to the variability in DNA damage carried out. Only for one tank, the measured electric field strength coincided with the expected 1 V/cm, while for four it ranged from 0.6 to 2.0 V/cm. The values of DNA damage assessed in the same samples of mouse kidney cells differed between tanks up to 4.7-fold for induced and up to 10-fold for spontaneous DNA damage. High local variations in the electric field strength and solution temperature across the platform as well as in %DNA in the tail of identical cell samples within electrophoresis runs also revealed. These variations were reduced by recirculation of electrophoresis solution. The results show that discrepancy between the estimated and the actual electric field strength can be reason of inter-laboratory variation of the comet assay results. Recirculation of the solution during electrophoresis will be useful to control of intra-laboratory and intra-assay variations.

About the authors

Aliy K. Zhanataev

Zakusov Research Institute of Pharmacology

Author for correspondence.
Email: zhanataev@academpharm.ru
ORCID iD: 0000-0002-7673-8672
SPIN-code: 7070-0510
Scopus Author ID: 6506103462

PhD, Main Researcher, Laboratory of Pharmacology and Mutagenesis

Russian Federation, Moscow

Elena A. Anisina

Zakusov Research Institute of Pharmacology

Email: anisinalena@yandex.ru

Researcher, Laboratory of Pharmacology and Mutagenesis

Russian Federation, Moscow

Kira L. Pligina

Zakusov Research Institute of Pharmacology

Email: kira-pligina@rambler.ru
SPIN-code: 7315-5417

PhD, Researcher, Laboratory of pharmacology and mutagenesis

Russian Federation, Moscow

Artem A. Lisitsyn

Zakusov Research Institute of Pharmacology

Email: nordikal@yandex.ru

Researcher, Laboratory of pharmacology and mutagenesis

Russian Federation, Moscow

Andrey D. Durnev

Zakusov Research Institute of Pharmacology

Email: addurnev@mail.ru
ORCID iD: 0000-0003-0218-8580
SPIN-code: 8426-0380
Scopus Author ID: 7006060753

Doctor of Science, Professor, Corresponding member of Russian Academy of Sciences, Head of Institute

Russian Federation, Moscow

References

  1. Koppen G, Azqueta A, Pourrut B, et al. The next three decades of the comet assay: a report of the 11th international comet assay workshop. Mutagenesis. 2017;32(3):397-408. https://doi.org/10.1093/mutage/gex002.
  2. Møller P. The comet assay: ready for 30 more years. Mutagenesis. 2018;33(1):1-7. https://doi.org/10.1093/mutage/gex046.
  3. Azqueta A, Gutzkow KB, Brunborg G, Collins AR. Towards a more reliable comet assay: optimizing agarose concentration, unwinding time and electrophoresis conditions. Mutation Research. 2011;724(1-2):41-45. https://doi.org/10.1016/j.mrgentox.2011.05.010.
  4. Azqueta A, Muruzabal D, Boutet-Robinet E, et al. Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies. Mutat Res. 2019;843:24-32. https://doi.org/10.1016/j.mrgentox.2019.04.007.
  5. Forchhammer L, Johansson C, Loft S, et al. Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial. Mutagenesis. 2010;25(2):113-123. https://doi.org/10.1093/mutage/gep048.
  6. Johansson C, Møller P, Forchhammer L, et al. An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay. Mutagenesis. 2010;25(2):125-132. https://doi.org/10.1093/mutage/gep055.
  7. Ersson C, Möller L. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments. Mutagenesis. 2011;26(6):689-695. https://doi.org/10.1093/mutage/ger034.
  8. Speit G, Trenz K, Schütz P, et al. The influence of temperature during alkaline treatment and electrophoresis on results obtained with the comet assay. Toxicol Lett. 1999;110(1-2):73-78. https://doi.org/10.1016/S0378-4274(99)00137-X.
  9. Sirota NP, Zhanataev AK, Kuznetsova EA, et al. Some causes of inter-laboratory variation in the results of comet assay. Mutat Res Genet Toxicol Environ Mutagen. 2014;770:16-22. https://doi.org/10.1016/j.mrgentox.2014.05.003.
  10. Møller P, Möller L, Godschalk RW, Jones GD. Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay validation group. Mutagenesis. 2010;25(2):109-111. https://doi.org/10.1093/mutage/gep067.
  11. Godschalk RW, Ersson C, Riso P, et al. DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay validation group (ECVAG). Mutat Res. 2013;757(1):60-67. https://doi.org/10.1016/j.mrgentox.2013.06.020.
  12. Ersson C, Møller P, Forchhammer L, et al. An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells. Mutagenesis. 2013;28(3):279-286. https://doi.org/10.1093/mutage/get001.
  13. Plappert-Helbig U, Guérard M. Inter-laboratory comparison of the in vivo comet assay including three image analysis systems. Environ Mol Mutagen. 2015;56(9):788-793. https://doi.org/10.1002/em.21964.
  14. Olive PL, Wlodek D, Durand RE, Banáth JP. Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp Cell Res. 1992;198(2):259-267. https://doi.org/10.1016/0014-4827(92)90378-l.
  15. Brunborg G, Rolstadaas L, Gutzkow KB. Electrophoresis in the comet assay. (September 12th 2018). In: Bolduna OM, Balta C, eds. Electrophoresis – Life Sciences Practical Applications. London: Intech Open; 2018. Р. 63-80. https://doi.org/10.5772/intechopen.76880.
  16. Sambrook J, Russel DW. Molecular cloning – a laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001.
  17. Olive PL, Banáth JP. The comet assay: a method to measure DNA damage in individual cells. Nat Protoc. 2006;1(1):23-29. https://doi.org/10.1038/nprot.2006.5.
  18. OECD. Test No. 489: In vivo mammalian alkaline comet assay [Internet]. Paris: OECD Publishing; 2016. Available from: https://www.oecd.org/env/test-no-489-in-vivo-mammalian-alkaline-comet-assay-9789264264885-en.htm.
  19. Gutzkow KB, Langleite TM, Meier S, et al. High-throughput comet assay using 96 minigels. Mutagenesis. 2013;28(3):333-340. https://doi.org/10.1093/mutage/get012.
  20. Tice RR, Agurell E, Anderson D, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35(3):206-221. https://doi.org/10.1002/(SICI)1098-2280(2000)35:33.0.CO;2-J.
  21. Collins AR, Oscoz AA, Brunborg G, et al. The comet assay: topical issues. Mutagenesis. 2008;23(3):143-151. https://doi.org/10.1093/mutage/gem051.
  22. Collins AR, El Yamani N, Lorenzo Y, et al. Controlling variation in the comet assay. Front Genet. 2014;5:359. https://doi.org/10.3389/fgene.2014.00359.
  23. Roy M, Hamel A, Cardoso R. Effect of slide positioning in electrophoresis chamber over comet assay results [Internet]. Charles River Laboratories, Senneville, Quebec, Canada, H9X 3R3. Available from: https://www.criver.com/sites/default/files/resources/EffectofSlidePositioninginElectrophoresisChamberOverCometAssayResults.pdf.
  24. Brody JR, Kern SE. Sodium boric acid: a tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques. 2004;36(2):214-216. https://doi.org/10.2144/04362BM02.
  25. O’Conner JL, Wade MF, Zhou Y. Control of buffer pH during agarose gel electrophoresis of glyoxylated RNA. Biotechniques. 1991;10(3):300-302.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Geometric parameters of chambers, location of slides with cells on the platform and sections (indicated by digits 1–12) for determining the local electric field strength and temperature of electrophoresis solution in the CSL–COM40, Sub-Cell 192, and multiSUB Screen 32 chambers. M – interelectrode distance; H – height, L – length, W – width of the chamber platform

Download (62KB)
Indexing metadata
3. Fig. 2. COMPAC-50 chamber with vertical orientation of slides. 1–9 sections for determining the electric field strength. M – interelectrode distance; H – height of the solution for electrophoresis, L – length, W – width of the chamber reservoir

Download (320KB)
Indexing metadata
4. Fig. 3. DNA comets' morphology at different electric field strength (A, B, C, D; electrophoresis 20 minutes) and at 2-times increased of recirculation speed (E, F; Multi SUB Screen 32 tank). Bar scale – 50 µm

Download (212KB)
Indexing metadata

Copyright (c) 2020 Zhanataev A.K., Anisina E.A., Pligina K.L., Lisitsyn A.A., Durnev A.D.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
 


This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies