Immobilon ny charged nylon membrane

Total cellular RNA including viral RNA was isolated with Trizol and 25 μg of the isolated RNA was resolved in a 1.5% agarose gel containing 2.2 M formaldehyde. The RNA in the gel was transferred onto IMMOBILON NY+ charged nylon membrane (Milipore) for Northern blot analysis. RNAs on the sheet were detected with specific DIG-labeled probe using a full-length HBV-DNA template as described previously [45 (link)]. The RNA was detected with the DIG DNA labeling and detection kit (Roche Diagnostics, Basel, Switzerland) according to the instructions. The levels of reference gene mRNAs in total RNA were quantified by quantitative PCR as described below.

Viral DNA was isolated from intracellular viral capsids and detected with specific DIG-labeled probe as described previously [10 (link)]. In brief, to isolate the viral DNA, homogenized mouse livers or cells transfected with the HBV plasmid were subjected to an overnight sodium dodecyl sulfate-proteinase K digestion followed by phenol extraction and ethanol precipitation. 20 μg of the isolated DNA was separated in a 1% agarose gel, transferred onto IMMOBILON NY+ charged nylon membrane (Milipore) and detected with a HBV-specific DIG-labeled probe using a full-length HBV-DNA template. HBV-DNA-specific signals were quantified using Image J, and plasmid DNA signal was used as reference.