3. Early protocols for electrophoresis in the comet assay

Publications during the last 30 years specify experimental protocols often by referring to the early comet assay protocols. Quite often, however, the conditions are still undefined or confusing. This relates, in particular, to a lack of precise definition of the local electric potential; at the same time, there is a frequent—and surprising—specification of the current used (mostly 300 mA). We have attempted to understand the basis for the apparent misconceptions; it seems to reflect history rather than science. The electrophoresis step was described and specified somewhat differently in the three early publications [1–3]. Östling and Johanson [1] separated cellular DNA under neutral (non-alkaline) conditions, "… and electric potential of about 5 V/cm is applied for 5 minutes …". The tank size, the current or the volume of liquid are not specified, which is in fact quite OK since the magnitude of the local electric potential is defined. Singh and co-workers, the first to describe an alkaline version of the assay, specify that electrophoresis is carried out for 20 mins at 25 V, at 300 mA, and a depth of liquid of 0.25 cm [2]. Since the tank dimensions are not known, the local electric potential is undefined. In a follow-up review paper, the electrophoresis is at 12 V and 100 mA, with the circulation of the electrophoresis solution at 100 ml/min of a total volume of 1 liter [10]; again, this implies an undefined electric potential. In a later publication [11], the same group describes electrophoresis at 12 V (0.4 V/cm), 250 mA, and a duration of 20 mins.

4. Results

Table 1.

potential [17].

4.1. Electrophoresis conditions and DNA damage levels

We have previously studied experimentally [17] the conditions which determine the level of migration measured in the comet assay. Cell cultures of two types were treated with genotoxicants and then assayed for DNA damage using different physical conditions during electrophoresis. Linear regression of the experimental data in [17] shows highly linear relationships for V/cm and time (Table 1) and an inverse relationship for agarose. Fitting straight lines to the figures in [17] results in the regression data (for human blood mononuclear cells) in

There was no effect of altering the electrophoresis current (between 210 and 400 mA), except for a slight downward trend which is explained as an indirect effect on the electric

Since the local electric potential determines the mobility of DNA fragments in the comet assay, it is essential that the electric potential is defined at all positions of the electrophoresis platform. This may be particularly important if the comet gels are small in the surface. We have described a high-throughput comet assay with 96 minigels, each of 4 μl, placed in an array of Gelbond plastic membranes as substrate [18, 19]. The minigels give results which are highly similar to results obtained with standard glass slides. We also evaluated this revised comet assay with respect to variation in DNA damage levels measured within one electrphoresis. Based on preliminary studies [18], DNA damage measured in parallel samples depended significantly on the circulation of the liquid during electrophoresis. We now report on a series of measurements of local electric potentials, with the purpose of identifying possible timedependent differences in electrophoresis between neighboring samples. A multi-electrode gauge was made (Figure 1) consisting of 20 evenly spaced (5 mm) platinum electrodes, each

This multi-electrode gauge was placed in an electrophoresis tank with the free electrode ends immersed in a thin layer of agarose of the same concentration (0.675%) as used to embed cells in the comet assay (Figure 2) [20]. The electrode ends were a fraction of a millimeter above the

Voltage (V/cm) (0.16–1.48) 34.1 (\*V/cm) 0.964 Linear, through zero Time (min) (5–40) 1.42 (\*min) 0.950 Linear, through zero

Table 1. Linear regression of data from [17], with goodness-of-fit. The regression curves for electric potential (V/cm) and

Agarose (%) (0.4–0.95) 30.29 (\*% + 52.7) 0.955 Inverse

) Comment

Electrophoresis in the Comet Assay

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http://dx.doi.org/10.5772/intechopen.76880

4.2. Position- and time-dependent variations in local electric potential

covered with a thin plastic tube except for 1 mm protruding free end.

Factor and unit (and range) Linear regression parameters Goodness-of-fit (R2

time (min) were both forced through zero.

In guidelines from 2000 [12], R.T. Tice (who was also co-author of the 1988 protocol with N. P. Singh [2]) states that "… due to the large variability in the size of commercially available electrophoresis units, it is more accurate and useful to present the voltage in V/cm". Concerning voltage, 0.7–1.0 V/cm and a duration of 5–40 mins are specified, and circulation (whereas current, liquid volume, and depth are not mentioned in these guidelines) [12]. In the comet assay later described by Peggy Olive [3, 13], electrophoresis was run at 1 V/cm for 20 min (nothing said about volume and current). In a follow-up protocol by Peggy Olive in 2006 [14], electrophoresis is conducted at 0.6 V/cm for 25 min; "The current should be about 40 mA if using 20 V. The distance in centimeters is measured between the negative and positive electrophoresis in the electrophoresis chamber." There is a problem associated with this approach, which we will discuss later.

Among the early comet assay protocols, the most cited ones (by March 2018) are [2] (6522 citations) and [12] (2906 citations); citations for Östling & Johansson [1] are not available. Judging from these bibliographic data, and since the electrophoresis conditions in [2] are undefined with respect to electric potential but recommending a specific current (300 mA), it seems that much of the confusion in the literature stems from the uncritical reference to [2]. A more frequent use of the guidelines [12] might have avoided some of the unexpected comet assay results published during the last 20–30 years.

The current plays no direct role in electrophoretic mobility, but may indirectly affect the local distribution of the electric potential in a tank. The organisation for economic co-operation and development (OECD) In vivo comet assay Test Guidelines protocol 489 [15] from 2014, correctly states that the "… level of DNA migration is linearly associated with the duration of electrophoresis, and also with the electric potential (V/cm)." However, there is still the recommendation of a "starting current of 300 mA (—), the depth should be adjusted to achieve these conditions, and the current at the start and the end of the electrophoresis should be recorded". It is worth noticing, however, that attempts to control the current often lead to very shallow liquid levels. In [16] the level of the buffer is described as "… about 2–3 mm above the agarose on the slide"; in [2] the depth was 0.25 cm. It is often underlined in the protocols that the electrophoresis tank must be in the level, so that the depth above the gels is equal over the platform surface. However, keeping a large tank level in each run, within an error of, for example, 1 mm is hardly achievable. The implication is that the resistance of the liquid on the platform will vary relatively much, leading to potentially large differences in local electric potential. Specifying a much higher depth, for example, 6–10 mm solves this problem, although a larger current would put demands on the power supply. We will get back to this problem in the recommendations at the end of this text.
