Application of digital PCR for assessing DNA fragmentation in cytotoxicity response

Abstract

Background: Regulated cell death plays an essential role in various biological processes, leading to the development of a number of methods to detect and quantitatively measure cells exhibiting decreased viability due to either apoptosis or necrosis. Methods and results: When cytotoxicity is induced by anti-cancer chemicals, human cell lines exhibit specific features, including dampened cell proliferation and lost plasma membrane asymmetry, presenting distinct sensitivity. In this study, we report a set of novel digital PCR (dPCR) assays to quantitatively measure the degree of cell death. These dPCR assays are designed to quantify targets of increasing sizes within the RNase P (RP) gene locus. The ratio between short and long target copy numbers implies the degree of DNA fragmentation, which we name the RP fragmentation index. Conclusions: Compared to other conventional quantitative methods, the RP fragmentation index using cellular DNA represents a valid indicator in the measurement of the degree of cell death. General significance: The demonstrated dPCR assays can precisely assess DNA fragmentation that quantitatively reflects the degree of cytotoxicity.