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Application of digital PCR for assessing DNA fragmentation in cytotoxicity response
- Han, Jeongran, Lee, Ji Youn, Bae, Young-Kyung
- Biochimica et biophysica acta 2019 v.1863 no.8 pp. 1235-1242
- DNA, DNA fragmentation, apoptosis, asymmetry, cell proliferation, cytotoxicity, genes, human cell lines, loci, plasma membrane, polymerase chain reaction, quantitative analysis, ribonucleases, viability
- 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.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.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.The demonstrated dPCR assays can precisely assess DNA fragmentation that quantitatively reflects the degree of cytotoxicity.