5. Discussion and conclusions

The ultimate aim of this study [20] was to establish electrophoresis conditions that are optimal for the comet assay, hopefully contributing to reducing the unexplainable variations, which are often found both within and between experiments. A further aim is to establish correction

Figure 7. Dose-response curves showing DNA damage versus radiation dose. One experiment; scoring of DNA damage in 96 samples with the semi-automated (Perceptives) and the automated scoring (IMSTAR Pathfinder) systems. Irradiation with X-rays (0–15 Gy) and circulation (109 ml/min) during electrophoresis. The Tail%DNA relative to the head is given as the median value of at least 50 comets in each sample. Linear regression lines are shown, with linear relations y = 3.99 + 4.37x and y = 0.1 + 4.30x, and coefficients of variation (CV) R<sup>2</sup> = 0.72 and R<sup>2</sup> = 0.96, for IMSTAR and Perceptives, respectively. The experimental points at dose 15 Gy were omitted from the linear regression.


rank sum test—not requiring the normal distribution of samples—was used when the Tail%

Figure 6. Average electric potentials measured in several experiments. Time-integrated corrected voltage per cm averaged for all electrodes during an electrophoresis time of 25 mins for each measurement with (109 ml/min, lower panel) and without (upper panel) circulation. The standard deviations are shown as red error bars and illustrate the degree of

We observed small differences in Tail%DNA levels in experiments using different rates of circulation. This is illustrated in Table 3 and in Figure 9, both based on weighted data (to

The ultimate aim of this study [20] was to establish electrophoresis conditions that are optimal for the comet assay, hopefully contributing to reducing the unexplainable variations, which are often found both within and between experiments. A further aim is to establish correction

DNA values were shown not to be normally distributed by the Lilliefors test.

variation in voltage per cm for each measurement. Figure from [20].

avoid that low numbers of scored comets should be given too much weight).

5. Discussion and conclusions

72 Electrophoresis - Life Sciences Practical Applications

Figure 8. Illustration of positioning of Gelbond films with one film (left; 96 minigels), or four films (right; 384 minigels) on a platform in an electrophoresis tank. Figure from [20].


be due to a better stabilization of local temperature that could alter the conductivity; a further mechanism could be a build-up of local concentration gradients of electrophoresis liquid, which are likely to be reduced by an increased flow. A positive feedback loop could then possibly take

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The logical extension of these observations was that the variations should be paralleled by variations in the level of DNA damage. We therefore examined populations of cells exposed to a genotoxic treatment (X-rays) under different electrophoresis conditions. The mean Tail% DNA of all samples in our minigel 4 96 array was remarkably constant, as concluded from three independent experiments (Figure 9). There was, however, a slight—but statistically not significant—tendency to a lower CV at the two highest rates of circulation (Table 3). This was unexpected in view of the markedly reduced variations in local electric potential with circula-

A statistical analysis of Tail%DNA of samples in the row and column positions in the 4 96 array also did not reveal systematic differences that could be attributed to electric potential variations. However, for each film, there were clear indications of lower levels of median DNA damage in samples, which were applied late in each pipetting (data not shown). We interpret this as due to some rapid repair of radiation damage, during the short period when cell

It is possible that variations in electric potentials are related to properties of the measuring gauge. The electric potential is dependent on the concentration gradient in a solution. Small differences in the concentration of reactants and products involved in the redox reaction

Figure 9. Mean Tail%DNA in electrophoresed samples of cells irradiated with X-rays (8 Gy). DNA damage levels of data from three experiments are shown (+/ STD; error bars) as a function of circulation flow rate. Figure from [20].

place in the absence of circulation.

tion. This is also unlike our preliminary findings [18].

samples are heated to 37C for mixing with agarose.

Table 3. Mean Tail%DNA in electrophoresed samples of cells irradiated with X-rays (8 Gy). Standard deviations of parallel samples and coefficients of variation are also listed. Data are from three experiments with or without circulation at different flow rates during electrophoresis. Data are weighted on the basis of the number of comets scored in each sample.

factors, which may be used to translate data from laboratories using different electrophoresis conditions.

A systematic investigation of comet assay electrophoresis had not previously been carried out. Since the electric potential is known to be the major physical force causing charged molecules to move during electrophoresis, we recorded the electric potential at multiple sites on the platform of a standard horizontal tank used for comet assay electrophoresis. In a previous study, we had observed that variations in DNA damage levels among parallel samples were considerably reduced when the liquid was circulated during electrophoresis [18].

We made detailed measurements of electric potentials using a gauge containing 20 platinum electrodes coupled to a voltage recording device with input resistance in the mega-ohm range. Using this setup we could confirm that the measured local electric potential was highly affected by circulating the electrophoresis liquid; that is, time- and position- dependent variations seemed to be reduced by circulation. For a standard electrophoresis time of 25 mins, the time-integrated variation (Coefficient of Variation, CV) of the electric potential at electrode positions was more than 8% without circulation; this was reduced to less than 1% with circulation at more than 58 ml/min, i.e. 5% volume change per min (Table 4). We initially hypothesized this reduction to


Table 4. Electric potentials and their variation at electrode positions as a function of circulation flow rate. The corrected electrode voltage per cm averaged over 25 mins is shown, with CV averaged for all electrodes. Data from three experiments with and without circulation at different flow rates.

be due to a better stabilization of local temperature that could alter the conductivity; a further mechanism could be a build-up of local concentration gradients of electrophoresis liquid, which are likely to be reduced by an increased flow. A positive feedback loop could then possibly take place in the absence of circulation.

The logical extension of these observations was that the variations should be paralleled by variations in the level of DNA damage. We therefore examined populations of cells exposed to a genotoxic treatment (X-rays) under different electrophoresis conditions. The mean Tail% DNA of all samples in our minigel 4 96 array was remarkably constant, as concluded from three independent experiments (Figure 9). There was, however, a slight—but statistically not significant—tendency to a lower CV at the two highest rates of circulation (Table 3). This was unexpected in view of the markedly reduced variations in local electric potential with circulation. This is also unlike our preliminary findings [18].

A statistical analysis of Tail%DNA of samples in the row and column positions in the 4 96 array also did not reveal systematic differences that could be attributed to electric potential variations. However, for each film, there were clear indications of lower levels of median DNA damage in samples, which were applied late in each pipetting (data not shown). We interpret this as due to some rapid repair of radiation damage, during the short period when cell samples are heated to 37C for mixing with agarose.

factors, which may be used to translate data from laboratories using different electrophoresis

Table 3. Mean Tail%DNA in electrophoresed samples of cells irradiated with X-rays (8 Gy). Standard deviations of parallel samples and coefficients of variation are also listed. Data are from three experiments with or without circulation at different flow rates during electrophoresis. Data are weighted on the basis of the number of comets scored in each

Flow rate (ml/min) Mean tail DNA intensity (%) Mean STD (%) Mean CV (%)

 39.53 4.15 10.50 42.75 4.31 10.07 34.83 3.89 11.18 34.75 3.28 9.44 36.27 2.28 8.14

A systematic investigation of comet assay electrophoresis had not previously been carried out. Since the electric potential is known to be the major physical force causing charged molecules to move during electrophoresis, we recorded the electric potential at multiple sites on the platform of a standard horizontal tank used for comet assay electrophoresis. In a previous study, we had observed that variations in DNA damage levels among parallel samples were

We made detailed measurements of electric potentials using a gauge containing 20 platinum electrodes coupled to a voltage recording device with input resistance in the mega-ohm range. Using this setup we could confirm that the measured local electric potential was highly affected by circulating the electrophoresis liquid; that is, time- and position- dependent variations seemed to be reduced by circulation. For a standard electrophoresis time of 25 mins, the time-integrated variation (Coefficient of Variation, CV) of the electric potential at electrode positions was more than 8% without circulation; this was reduced to less than 1% with circulation at more than 58 ml/min, i.e. 5% volume change per min (Table 4). We initially hypothesized this reduction to

Table 4. Electric potentials and their variation at electrode positions as a function of circulation flow rate. The corrected electrode voltage per cm averaged over 25 mins is shown, with CV averaged for all electrodes. Data from three

considerably reduced when the liquid was circulated during electrophoresis [18].

Flow rate Mean corrected electrode voltage Mean CV

ml/min V/cm % 0.82 8.28 0.85 1.17 0.86 0.53 0.83 0.65 0.84 0.70 0.83 0.50

experiments with and without circulation at different flow rates.

conditions.

74 Electrophoresis - Life Sciences Practical Applications

sample.

It is possible that variations in electric potentials are related to properties of the measuring gauge. The electric potential is dependent on the concentration gradient in a solution. Small differences in the concentration of reactants and products involved in the redox reaction

Figure 9. Mean Tail%DNA in electrophoresed samples of cells irradiated with X-rays (8 Gy). DNA damage levels of data from three experiments are shown (+/ STD; error bars) as a function of circulation flow rate. Figure from [20].

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

typical voltage for an electrophoresis chamber is determined by measuring the perpendicular distance in cm between the positive and negative electrodes in the electrophoresis chamber, and multiplying this distance by 0.6." This simplified method to determine the electric potential (V/cm) may introduce errors and should not be used. Firstly, there is a voltage drop at each electrode (the electrode potential at standard state for platinum equals 1.18 V [22]). This electrode potential is evident from the measurement of the current at increasing electrode voltage (from the power supply) in a tank; see Figure 10. There is no current at voltages lower than 2.5 V; the curve extrapolates at zero current to 2.7 V, which is close to the electrode potentials at both electrodes (2.38 V). Furthermore, the side wells have higher conductivity (per length) than the platform. In a small tank, the electrode potential may

Care should also be taken concerning the resistance of the cathode and anode electrodes. There is a voltage drop along these electrodes, even with platinum (specific resistance 1.05 \* 10<sup>7</sup>

\*m (at 20C)). Depending on the electrode cross-section and the current, this could result in as much as 5–10% lower voltage at the far end of the tank (i.e. opposite to where the connecting leads are). This error can be reduced by using thicker (very expensive) platinum, but a cheaper solution is to connect both ends of each electrode to the power supply (4 leads). An alternative approach is to measure and/or calculate the electric potential (V/cm) along both sides of the platform and introduce correction factors to account for the difference in each position.

In summary, based on our experiments and the evaluations above, we suggest a number of

• When introducing a new tank: measure and record the voltage and the current of the

• Circulate the electrophoresis liquid during electrophoresis using an external pump (e.g., there are cheap peristaltic pumps made for aquariums). The advantages are (1) stable conditions allowing more precise measurement of the electric potential; (2) more stable temperature during electrophoresis; and (3) (probably) reduced variations in the local

• Use sufficient volume of electrophoresis liquid to cover the agarose samples with at least

• Ensure that a power supply is used which can supply the output current at the constant voltage and with sufficient volume of liquid (two car batteries are a very cheap and useful

• Use electrodes of sufficient size (cross section), to avoid a voltage drop due to high ohmic resistance. The overall resistance is reduced if both ends of each electrode are connected to

6. Recommendations for comet assay electrophoresis

electrophoresis tank, when containing different volumes of liquid.

ohm

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introduce serious errors.

simple recommendations:

electric potential.

5 mm.

alternative).

the output cable from the power supply.

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

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

typical voltage for an electrophoresis chamber is determined by measuring the perpendicular distance in cm between the positive and negative electrodes in the electrophoresis chamber, and multiplying this distance by 0.6." This simplified method to determine the electric potential (V/cm) may introduce errors and should not be used. Firstly, there is a voltage drop at each electrode (the electrode potential at standard state for platinum equals 1.18 V [22]). This electrode potential is evident from the measurement of the current at increasing electrode voltage (from the power supply) in a tank; see Figure 10. There is no current at voltages lower than 2.5 V; the curve extrapolates at zero current to 2.7 V, which is close to the electrode potentials at both electrodes (2.38 V). Furthermore, the side wells have higher conductivity (per length) than the platform. In a small tank, the electrode potential may introduce serious errors.

Care should also be taken concerning the resistance of the cathode and anode electrodes. There is a voltage drop along these electrodes, even with platinum (specific resistance 1.05 \* 10<sup>7</sup> ohm \*m (at 20C)). Depending on the electrode cross-section and the current, this could result in as much as 5–10% lower voltage at the far end of the tank (i.e. opposite to where the connecting leads are). This error can be reduced by using thicker (very expensive) platinum, but a cheaper solution is to connect both ends of each electrode to the power supply (4 leads). An alternative approach is to measure and/or calculate the electric potential (V/cm) along both sides of the platform and introduce correction factors to account for the difference in each position.

In summary, based on our experiments and the evaluations above, we suggest a number of simple recommendations:
