Nonfat skim milk

Total RNAs were quantified using Nanodrop (Thermo), mixed to 6X DNA loading buffer (Invitrogen) and then resolved on 3.5% non-denaturing polyacrylamide gels. Samples were wet transferred to a positively charged nylon membrane (GE) in TBE 0.5X at 35V/4°C/overnight. The RNAs were crosslinked to the membrane using 120 mJ/cm² UVC (Stratagene). Membranes were washed in 1X PBS and incubated in the blocking buffer consisted of PBST (1X PBS and 0.1% Tween-20), 5% of non-fat skim milk (Bio-Rad), 50 μg/mL sheared salmon DNA. After 1 hour shaking at RT, blocking buffer was replaced for the primary antibody solution consisting of PBST, 2% non-fat skim milk and 1/6000 of recombinant rabbit anti-dsRNA 9D5 antibody (Absolute antibody). Membranes were incubated for 2 hours shaking at RT, washed with PBST and incubated for 1 hour shaking at RT in the 1/10000 of HPR-linked anti-rabbit antibody (Amersham) solution (1X PBST, 2% non-fat skim milk). The membranes were washed with PBST and rinsed in 1X PBS. Then, the ECL substrate (GE) was added to blots according to the manufacturer’s recommendations. Chemiluminescent signals were acquired using a Fusion FX7 Image System. Data were analyzed with ImageJ software. dsRNA sizes were estimated based on the pattern of purified dsRNAs from DCV-infected cells in an 1% agarose gel.