For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.
Comprehensive HBV Nucleic Acid Extraction
For HBV RNA Northern blot analysis, total cellular RNA or encapsidated pgRNA samples were resolved in a 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond-XL membrane (GE Healthcare). For DNA Southern blot analysis, HBV core DNA or cccDNA samples were resolved by electrophoresis in a 1.2% agarose gel and blotted onto a Hybond-XL membrane. Membranes were probed with either an [α-32P]UTP (3,000 Ci/mmol; PerkinElmer)-labeled plus-strand-specific (for Northern blot hybridization) or minus-strand-specific (for Southern blot hybridization) HBV riboprobe and exposed to a phosphorimager screen. Extracellular HBV DNA and RNA were copurified from the culture medium of induced HepAD38 cells by using a MinElute virus vacuum kit (Qiagen). Viral DNA was quantified using the above-mentioned qPCR method directly, and HBV RNA was detected by RT-qPCR with the same core region primer/probe set after the removal of DNA contamination by DNase I (Thermo Fisher) treatment.
Variable analysis
- Total cellular RNA
- Cytoplasmic encapsidated HBV pgRNA
- Core DNA
- Protein-free Hirt DNA
- HBV RNA Northern blot analysis
- HBV DNA Southern blot analysis
- Extracellular HBV DNA
- Extracellular HBV RNA
- Non-cccDNA in Hirt DNA was removed via T5 exonuclease digestion
- EcoRI endonuclease was used to linearize HBV cccDNA before electrophoresis
- None specified
- None specified
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