An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells

Abstract

Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. For complete details on the use and execution of this protocol, please refer to Jahun et al.¹.

Keywords: Cancer; Cell Biology; Cell separation/fractionation; Cell-based Assays; Immunology; Molecular Biology.

Graphical abstract

Figure 1

Representative results showing leakage of DNA into the cytosol in MNV1-infected cells (A) Schematic representation of the workflow. (B‒H) HEK293T cells stably expressing the mouse CD300lf receptor were infected with MNV1 in 3 replicates at an MOI of 0.05 TCID50/cell. Cells were harvested 18 h post-infection and cytosolic DNA was assessed relative to mock-infected cells. Whole cell lysates were used for western blotting for MNV1 NS7 and GAPDH, to show virus infection (B). Panels (C) to (H) show levels of indicated cytosolic DNA in infected cells normalized to individual whole cell DNA, relative to mock-infected conditions. Error bars represent standard error of mean. nuclear-DNA1 = 18S, nuclear-DNA2 = GAPDH, mtDNA1 = RNR2-TRNL1 junction, mtDNA2 = D-loop region, mtDNA3 = COX2, mtDNA4 = ATP6.