A novel approach to increase robustness, precision and high-throughput capacity of single cell gel electrophoresis

Abstract

Routine use of the 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 scientific know-ledge on the 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 the 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.
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.