Abstract
Abstract: :
Purpose: To measure the amount of DNA damages represented by the steady state level of single strand breaks (SSBs) and double strand breaks (DSBs) in a small volume of retinal pigment epithelial cells . Methods: Terminal deoxynucleotidyl transferase (TdT) mediated end labeling is used for detecting DSBs and SSBs. The sensitivity of TdT to detect SSBs is normally quite limited, but it may be significantly improved by first denaturing the double strands and expose all the DNA nicks as potential substrates for TdT, followed by slot blot southern hybridization. Results: SSBs as well as DSBs were quantitatively detected in 20ng DNA samples derived from a retinal pigment epithelial cell line treated with H2O2. The signal intensity of denatured and TdT–treated DNA in slot blot hybridization correlated to the amount of SSBs calculated in an S1 nuclease digestion assay. The signal ratio between denatured and non–denatured DNA likely approximates the SSBs/DSBs ratio in genomic DNA. Conclusions: The combination of DNA denaturing, TdT treatment, and slot blot hybridization could be a useful method to assess oxidative stress–induced DNA strand damages in a small volume of retinal pigment epitheial cell samples. The applicability of this method to in vivo sample will be investigated.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • detection