Hirt DNA extraction, Southern blot analysis, and real-time detection PCR (RTD-PCR) quantification of HBV cccDNA

The Hirt protein-free DNA extraction procedure was used to isolate HBV cccDNA from HBV-infected cells⁴⁴. HBV preS/S fragments were obtained by PCR amplification with the appropriate forward (5′-TTTTGAATTCATGGGAGGTTGGTCTTCCAAACC-3′) and reverse (5′-TTTTGCGGCCGCTCAAATGTATACCCAAAGACAAAAGA-3′) primers (TaqMan, Thermo Fisher Scientific, Waltham, MA). The amplified HBV preS/S fragments were inserted into a pSPT18 vector to generate a pSPT18-pres/s plasmid. The pSPT18-pres/s template was linearized by HindIII (Takara, Shiga, Japan) and in vitro transcription was performed with 1 µg linearized DNA template using a DIG RNA Labeling Kit (Roche, Basel, Switzerland) in the presence of T7 RNA polymerase to generate digoxigenin-UTP-labeled, single-stranded RNA probes. Hirt-extracted DNA was electrophoresed on 1.2% agarose gels and blotted onto a Hybond^TM-N + membrane (GE Healthcare, Amersham, UK). DIG-labeled single-stranded RNA probes transcribed from pSPT18-pres/s plasmids were used to detect HBV cccDNA. Hybridization was performed with a 30-min pre-hybridization at 50 °C in 10 mL of DIG Easy Hyb™ buffer (Roche) and with overnight hybridization at 50 °C in 3.5 mL of pre-warmed DIG Easy Hyb™ buffer containing 1 µg of DIG-labeled probe (Roche). The membranes were then washed with a Wash and Block Buffer Set (Roche) according to the manufacturer’s instructions. Probe-target hybrids were localized with 2 µL of anti-digoxigenin-AP conjugate (Roche) and detected by 1 mL CSPD (Roche). Images were acquired with the ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA). Hirt-extracted DNA was digested as previously reported⁴⁵. HBV cccDNA expression was quantified with TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) using a specific HBV cccDNA probe (5′-FAM-ACCACCGTGAACGCCCA-MGB-3′), forward primer (5′-GCGCACCTCTCTTTACGCG-3′), and reverse primer (5′-GCCCCAAAGCCACCCA AG-3′) (FASMAC, Kanagawa, Japan) as follows: 50 °C for 2 min, 95 °C for 10 min, and 45 cycles of 95 °C for 10 min and 65 °C for 30 s. HBV DNA and cccDNA copy number was quantified relative to a known copy number of HBV DNA.