The comet assay or single-cell gel electrophoresis (SCGE) has been widely used over a decade as a simple, rapid, and sensitive way for determining deoxyribonucleic acid (DNA) strand breaks. The versatility of the SCGE assay is because of the fact that a wide range of fresh or frozen samples can be used, including peripheral blood, cultured cells, buccal mucosal cells, solid tumours, cancer cells, yeast cells, epithelial cells, bacteria etc. The most extensively used form of this method is the alkaline version as it detects a variety of damages in single cells, such as DNA double-strand breaks, single-strand breaks, alkali-labile sites and DNA-DNA/DNA-protein cross-linking. Thus, the human epithelial bladder cancer (RT112) cell line was incubated with hydrogen peroxide (H2O2), a DNA-damaging agent. The cells were embedded in a thin agarose gel on microscope slides. Cellular proteins were removed by lysing the cells, followed by unwinding the damaged DNA-forming comets using electrophoresis under alkaline conditions (pH >13). One hundred comet images per each concentration were captured by using a fluorescence microscope. There was a significant increase in DNA percentage in tail of the comets with a linear rising of the tail lengths. Additionally, there was a strong positive correlation between tail DNA percentage and the lengths of the tail comets (p < 0.05, r2 > 0.98). Overall, the results of this study indicate that by increasing the DNA damage by H2O2, the intensity of the comet tails becomes higher, and the alkaline form of the comet assay is an effective method for measuring this damage.
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