Abstract
The routine use of single cell gel electrophoresis assay in medical diagnostics and biomonitoring is prevented by its high variability. Several factors have been identified and can be grouped into four main categories: 1) the biological sample, 2) the assay protocol, 3) the physical parameters during electrophoresis and 4) the analysis. Even though the scientific knowledge on assay variability is available, not much has been done so far to tackle the issues from the technological side. Therefore, this study addresses the question in how far the precise and accurate control over the physical parameters of electrophoresis is able to reduce variability of single cell gel electrophoresis assay results. All four above mentioned categories make up the overall assay variability. To resolve the contribution from a single category, the remaining three have to be kept as constant as possible. To achieve this we generated a set of x-ray treated control cells, worked according to a well-defined standard operating procedure and one single operator performed the analysis. Thereby variability resulting from the electrophoresis tank could be elucidated. We compared assay performance in two such tank systems: a newly developed electrophoresis tank that accurately controls voltage, temperature during the electrophoretic run and the homogeneity of the electric field, and a widely used commercially available standard platform tank. In summary, our results demonstrate that, irrespective of the cellular sample and its intrinsic biological variability, accurate control over physical parameters considerably increases repeatability, reproducibility and precision of single cell gel electrophoresis.
Highlights
Since its development in the 1980s (Ostling and Johanson, 1984; Singh et al, 1988), single cell gel electrophoresis (SCGE), otherwise known as the comet assay, has been applied to identify substances that cause DNA damage
The relatively simple method involves embedding the sample, i.e., a cell suspension retrieved from various sources such as cell cultures, liquid biopsies or dissociated tissue preparations, into low melting agarose (LMA) on glass slides
2.2 Cell culture A549 lung alveolar carcinoma (ATCC® CCL-185) cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal calf serum (FCS), 0.2 mM L-glutamine and 1 x penicillin, streptomycin, neomycin (PSN) (Gibco, Thermo Fisher Scientific, France), termed as complete cell culture medium, under standard growth conditions
Summary
Since its development in the 1980s (Ostling and Johanson, 1984; Singh et al, 1988), single cell gel electrophoresis (SCGE), otherwise known as the comet assay, has been applied to identify substances that cause DNA damage. It is an official test method for determining the genotoxic potential of any type of xenobiotic to which humans may be exposed (OECD, 2016) and has recently been used in medical settings such as the management of drug treatments in cancer patients (Apostolou et al, 2014).
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