Analysis of cf-mtDNA and cf-nDNA fragment size distribution using different isolation methods in BV-2 cell supernatant of starvation-induced autophagy

Fragment size distribution, the important biological properties of cell-free DNA (cfDNA), provides useful information required for diagnostic assay development. However, besides methodological discrepancies, it varies due to the complicated origins and occurrences of in vivo cfDNA. In addition, limited data are available concerning the cfDNA associated with autophagy and distributional difference between cf-mitochondrial DNA (cf-mtDNA) and cf-nuclear DNA (cf-nDNA) fragments. Here we developed an in vitro model of mouse microglial cell (BV-2) with starvation-induced autophagy, in which cfDNA was isolated from the cell supernatant by ultrafiltration (UF) and column-based commercial kit (CC), respectively. Using Agilent 2100 Bioanalyzer, a DNA ladder pattern as the presence of peaks corresponding to mono-, di- and tri-nucleosomes was clearly visualized both in isolation products of UF and CC. However, we also detected shorter fragments than mono-nucleosome by UF. In comparing the UF and CC, we found that the former produced the higher recovery efficiency for spiked-in DNA of shorter fragments than mono-nucleosome in both water and medium, but the latter was superior for spiked-in DNA fragments which were longer than or equal to mono-nucleosome in medium. Combined with these two isolation methods, we have observed that autophagy-associated cf-mtDNA and cf-nDNA were both highly enriched in

Automated summary

This study developed an in vitro model using mouse microglial cells to study autophagy-associated cfDNA, isolating cfDNA using ultrafiltration and a commercial kit. It was found that ultrafiltration had higher recovery efficiency for shorter DNA fragments, while the commercial kit was superior for longer DNA fragments.