single cell gel electrophoresis (the comet assay) continues to gain popularity as a mean of assessing dna damage. more and more researchers from universities, research institutes and related pharmaceutical companies have shown a strong interest in the comet assay. for example, the screening of genetic toxic drugs in pharmaceutical companies.
figure 1 | overview of the typical alkaline comet assay procedure.
whatever the comet assay is, it comes with the above several experimental steps. however, the assay’s low sample throughput and laborious sample workup procedure are limiting factors to its application. ''scoring'', or individually determining dna damage levels in 50 cells pretreatment is time-consuming in the traditional alkaline comet assay(aca).
in order to detect dna damage in large quantities of samples, a new high-throughput(ht) comet electrophoresis system compac-50 was developed by cleaver scientific and the oxidative stress group leicester university. the oxidative stress group of the university of leicester in a literature published in the scientific report detailing the upgrading process of the comet experiment device, referring to the great effect of the new ht rack and ht electrophoresis tank on the experiment. it also indicates that this upgrading greatly improves the number of samples to be analyzed, saves the experiment time, reduces the operation steps, reduces the reagent demand and the risk of breakage of glass slides. the compact design of electrophoresis tank is very useful for laboratories with small worktable space.
materials used in new methods:
simultaneous manipulation of up to 25 comet assay slides was achieved by using a polyoxymethylene rack, which was termed the ht rack (figure 2a). the same rack allowed electrophoresis to perform with the slides held, lengthwise, in a vertical orientation. although a custom-made electrophoresis tank (ht tank1; figure2b) was already available within our laboratory and proved suitably to demonstrate experiment principle,ht rack still has to be shortened to fit into the tank for operation. the tank design was then improved so as to accommodate two full size ht racks (named ht tank 2; manufactured by cleaver scientific ltd, rugby, uk), and used for all subsequent experiments involving further testing of the ht rack (figure 2c, right).
figure 2 | (a) representative illustration of the ht racks, which can accommodate up to 25 slides, and the staining dishes in which lysis, neutralisation, staining and all associated wash steps are performed. (figure 2a is reproduced here with the kind permission of comery, hill & cu., benthall, uk). (b) the ht tank 1 and ht rack. the ht tank 1 was used in preliminary, proof-of-principle experiments . (c) demonstration of the size difference between the conventional aca apparatus (left) and the ht tank 2 (right), which are separated by a power supply.
the paper also studied the effects on comet shape of performing electrophoresis on slides held vertically in the ht racks, the effect of buffer volume on tank voltage/current parameters, and the concentration-response and repeatability of ht comet assay.
it showed that the results obtained from the upgraded ht comet electrophoresis system are not significantly different from the aca, but the ht system decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. moreover, the risk of the gel breakage is significantly reduced.the cs-300v power supply (cs-300v; cleaver scientific ltd, rugby,uk) was proved to be perfectly suited to this application.
with the development of our products, cleaver scientific further improved the experimental device and then designed a suspended ht rack (figure 3a), and was equipped with a staining dish in the ht comet assay system to to deal with the slides in batches. at the same time, the ceramic cooling base (figure 3b) and the cooling pack are developed to ensure the optimum temperature of the electrophoresis buffer and improve the electrophoresis efficiency.
figure 3 | suspended ht rack and the ceramic cooling base