Dot Blot Analysis of DNA-RNA Hybrids

Dot blot analysis was performed with minor changes according to the protocol reported elsewhere (Morales et al., 2016 (link)). Genomic DNA was isolated by standard extraction with Phenol/Clorophorm/Isoamylic Alcohol (pH 8.0) followed by precipitation with 3 M NaOAc and 70% Ethanol. The isolated genomic DNA was randomly fragmented for 4 hr at 37 °C with a cocktail restriction enzymes (BsrgI, EcoRI, HindIII, XbaI, Ssp1) supplemented with 1 M spermidine. After incubation, the digested DNA was cleaned up with Phenol/Clorophorm extraction and standard Ethanol precipitation. After sample quantification, 350 ng of digested DNA was incubated with RNase H overnight at 37 °C as a negative control. The same quantity of each sample was spotted onto a nitrocellulose membrane, blocked in 5% non-fat dry milk, and incubated with the anti-DNA-RNA hybrid [S9.6] antibody (Kerafast; 1:1000) or anti-dsDNA antibody (Abcam; 1:2000) overnight at 4 °C. A horseradish peroxidase-conjugated goat specie-specific secondary antibody (Santa Cruz Biotechnology) was used. Quantification on the scanned image of the blot was performed using Image Lab software with dsDNA as loading control.