Mutation Research 724 (2011) 41–45 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis journa l h omepage: www.elsevier.com/locate/gentox C om mun i ty a ddress: www.elsevier.com/locate/mutres Towards a more reliable comet assay: Optimising agarose concentration, unwinding time and electrophoresis conditions Amaya Azqueta a,∗ , Kristine B. Gutzkow b , Gunnar Brunborg b , Andrew R Collins a a Department of Nutrition, Faculty of Medicine, University of Oslo, PB 1046 Blindern, 0316 Oslo, Norway b Department of Chemical Toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, PB 4404 Nydalen, 0403 Oslo, Norway a r t i c l e i n f o Article history: Received 14 January 2011 Received in revised form 19 April 2011 Accepted 8 May 2011 Available online 30 May 2011 Keywords: Comet assay Electrophoresis Alkaline unwinding Voltage Current Agarose concentration a b s t r a c t The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. How- ever, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, elec- trophoresis time, voltage and current, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Testing of chemicals for genotoxicity requires methods that are sensitive and specific, preferably economical and quick, and reliable. The alkaline (pH > 13) comet assay (single-cell gel elec- trophoresis) meets most of these requirements, but is regarded by some as unreliable. This reputation is not entirely deserved: it derives from the use of diverse conditions in different labo- ratories, and from a lack of understanding of the critical steps. Recent collaborations between laboratories that are experienced in the comet assay have highlighted variations in alkaline unwinding time (20–40 min), voltage gradient (0.76–1.6 V/cm), electrophore- sis time (20–30 min) and current (260–300 mA) [1,2]. Complete standardisation is not achievable, but in order to be able to com- pare results from different laboratories, it is important to identify the factors that affect performance of the assay. Here we examine specifically those factors that are expected to influence the outcome of electrophoresis: agarose concentration, alkaline incubation time, voltage, current, and time of electrophoresis. ∗ Corresponding author. Tel.: +47 22851360; fax: +47 22851341. E-mail addresses: o.a.azqueta@medisin.uio.no (A. Azqueta), kristine.bjerve.gutzkow@fhi.no (K.B. Gutzkow), gunnar.brunborg@fhi.no (G. Brunborg), a.r.collins@medisin.uio.no (A.R. Collins). In the comet assay, cells are embedded in agarose on a micro- scope slide or a plastic film, lysed, and electrophoresed in alkaline solution: DNA extends towards the anode, but only if strand breaks (or alkali-labile sites) are present, relaxing supercoiling. The % of DNA in the tail (estimated by fluorescence staining and image anal- ysis of the microscope images) reflects the frequency of breaks. Agarose concentrations of 0.5–1% are commonly reported. How- ever, the actual final concentration is often unknown and variable, since, in certain experimental designs, an aliquot of cells is cen- trifuged and the pellet re-suspended in an indeterminate volume before mixing it with agarose. The voltage set on the power supply is only a rough guide to the voltage applied across the platform carrying the gels, in electrophoresis tanks that vary considerably in size and design. A voltage gradient of 0.8–1 V/cm is often recommended, calculating this from the total voltage with the distance between electrodes as the divisor. But the main voltage drop occurs across the platform on which the slides are placed, where the layer of electrophoresis solution is at its thinnest (sometimes as little as a few mm above the slides) and the resistance greatest. There is little voltage drop over the width of the reservoirs on each side of the platform. To estimate the V/cm on the platform, one may divide the total applied voltage by the length of the platform, or – preferably – measure the actual voltage potential difference on the platform. In publications the current (in mA) is often specified, but this is unnecessary since the potential difference (V/cm) is the driving force in electrophoresis. If the same volume of solution is always used in a tank, the current should always be the same provided that 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.05.010