Detection of dsRNA and RNA:DNA Hybrids by Dot Blot Analysis

The IVT mRNA samples were diluted in nuclease-free water to final concentrations of 8, 40, 200, and 600 ng/μL, and 5-μL aliquots were then spotted to a positively charged nylon membrane (Whatman Nytran SuPerCharge, Sigma-Aldrich), resulting in a total amount of 40, 200, 1,000, and 3,000 ng IVT mRNA per dot, respectively. Before loading, the membrane was placed on a sheet of Whatman GB005 blotting paper and fixed with tape. Then a silicone mask (Bio-Dot Gasket 96 wells, Bio-Rad, Munich, Germany) was tightly pressed onto the membrane to ensure that all wells were sealed to avoid sample leakage. Loading was performed by pipetting 5 μL diluted sample into the wells of the silicone mask. This was achieved by placing the pipette tip vertically over the center of the well to ensure equal sample distribution on the area defined by the well. The sample liquid drained into the membrane and the underlying blotting paper by capillary forces, while the negatively charged RNA was captured by the positively charged nylon membrane. After loading, the sealing gasket was removed and the air-dried membrane transferred to a 50-mL tube and blocked in 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS)-T buffer (20 mM Tris [pH 7.4], 150 mM NaCl, and 0.1% (v/v) Tween-20).

For the detection of dsRNA, the membrane was incubated with J2 anti-dsRNA murine antibody (Scicons, Budapest, Hungary) diluted 1:5,000. To detect RNA:DNA hybrids, the incubation was performed with S9.6 murine monoclonal antibody (mAb) (Kerafast, Boston, MA, USA) diluted 1:20,000. The antibodies were diluted in 1% (w/v) non-fat dried milk and TBS-T and incubated with the membranes on a rolling mixer at 4°C overnight. The membranes were washed three times with 35 mL TBS-T for 15 min each and then incubated with horseradish peroxidase (HRP)-conjugated donkey anti-mouse immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK) diluted 1:10,000 in 1% (w/v) non-fat dried milk and TBS-T at room temperature for 1 h. After washing the membranes three times with 35 mL TBS-T for 15 min, chemiluminescence detection was performed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) and the ChemiDoc MP Imaging System (Bio-Rad). The signal intensities of the dots were quantified by densitometry using the Volume Tools of the Image Lab software (Bio-Rad). To verify equal sample loading, membranes were stripped with 1% (w/v) SDS containing 40 mM dithiothreitol at 60°C for 30 min and probed with a 3′-biotinylated oligodeoxynucleotide (5′-GTG AGT GGG GCA GGT GGA GGT GGG AGC ATA-3′) complementary to the 3′ UTR of the mRNA. Reprobing with oligodeoxynucleotide (ODN) was performed in PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich), according to the manufacturer, and HRP-conjugated streptavidin (Thermo Fisher Scientific) was used for visualization.