6. Recommendations for comet assay electrophoresis

occurring at each electrode surface during measurements may cause variation in the recorded electric potential. Circulating the electrophoresis solution may contribute to reducing the build-up of concentration differences, resulting in turn in less variation in the measured electric potential. A reference electrode should be used in order to determine the potential differences across the platform with higher precision. The reference electrode must have a well-defined and reproducible potential, that is, both reactants and products must be present with the kinetics of the reactions sufficiently fast for the species to be present at their equilibrium concentrations. Taken together, the measurements using the multi-electrode gauge were probably more representative for the local electric potential in the presence of

In conclusion, it seems that circulation of the liquid during electrophoresis has minor effects on the recorded level DNA damage. Even so, there are several arguments for using circulation in comet assay electrophoresis. Uncontrolled changes in temperature, not analyzed in this project but studied by Sirota and co-workers [21], may lead to heterogeneities in DNA damage expression. External peristaltic pumps combined with a spiral of stainless steel tubing placed in ice/water represent an efficient method to stabilize the temperature during electrophoresis, which also becomes more important with higher currents (more heat dissipation). Furthermore, standardization of data relies on the accurate determination of the electric potential. This should be determined using a suitable gauge; from our experiments, it appears that circulation stabilizes the measurement of the potential. It is stated in [16] that "A

circulation. Without circulation, however, these measurements are less reliable.

76 Electrophoresis - Life Sciences Practical Applications

Figure 10. Current in an electrophoresis tank measured as a function of the applied voltage.


• Measure the local potential on the platform, preferably using a gauge with platinum electrodes at defined positions. Measure in the presence of circulation.

[5] Forchhammer L, Johansson C, Loft S, Moller L, Godschalk RW, Langie SA, et al. Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-

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[7] Godschalk RW, Ersson C, Riso P, Porrini M, Langie SA, van Schooten FJ, et al. DNA-repair measurements by use of the modified comet assay: An inter-laboratory comparison within the European comet assay validation group (ECVAG). Mutation Research. 2013;

[8] Ersson C, Moller P, Forchhammer L, Loft S, Azqueta A, Godschalk RW, 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.

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