19, 20 Northern analysis was performed essentially as previously described.20 The RNAs were glyoxalated and then resolved using 1.7% agarose gels. The hybridization solution used was Ultrahyb Ultrasensitive Hybridization Buffer, containing 0.1 mg/mL of

sheared salmon sperm DNA (Applied Biosystems). The HDV RNAs were detected using 32P-labeled riboprobes, produced by in vitro transcription of either pTW108 or pTW107 plasmid linearized with HindIII. Plasmid Fulvestrant cell line pTW108 bears 1.1× unit-length HDV genome (G), whereas plasmid pTW107 contains 1.1× of the HDV antigenome (aG).20-22 Radioactivity was visualized using a biomolecular imager (Typhoon FLA 9000, GE Heathcare). Images were acquired and quantified using ImageQuant TL and prepared for publication using PowerPoint and Adobe Photoshop software. The procedure has been described in detail elsewhere.19 The primers used were: (1) forward primer, 312-GGACCCCTTCAGC GAACA-329; and (2) reverse primer, 393-CCTAGC ATCTCCTCCTATCGCTAT-360. The TaqMan probe was 332-AGGCGCTTCGAGCGGTAGGAGTAAGA-357.

The HDV numbering was according to Kuo et al.23 To assay G RNA, the above reverse primer was used in the reverse transcription (RT) reaction. To assay aG RNA, the above forward primer was used for the RT reaction. The copy numbers were quantified EPZ-6438 order using a 10-fold dilution series of either G or aG RNA standards (range: 20-200,000 GE of HDV). We deduce that 1 million HDV RNA molecules (G or aG) is equal to 1 pg of the corresponding HDV RNA standard. The 7500 Real Time PCR instrument (Applied Biosystems) was used for all qPCR assays according to the instructions of the manufacturer. The cccDNA-enriched fraction that is mostly free of cellular genomic DNA was isolated using previously described protocols24-26 and was further treated with RNase A (Roche), Hpa I (New England Biolabs), and Plasmid-Safe-ATP-dependent

DNase (Epicentre Technologies) to eliminate RNA, linear WHV DNA intermediates, double-stranded (ds) linear WHV genomic DNA, residual genomic cellular DNA, and certain forms of relaxed circular (rc) WHV DNA. The resulting DNA was subsequently treated with Mung Bean nuclease (New England Biolabs) to eliminate remaining relaxed circular DNA (rcDNA). The qPCR to quantify cccDNA assayed the dsDNA region unique for cccDNA. Forward primer, 1701-GGTCCGTGTT GCTTGGTCT-1719; reverse primer, 1977-GGACAT GGAACACAGGCAAAAACA-1954; this website and TaqMan probe, 1846-AATGGGAGGAGGGCAGCATTGATCCT-1871 were used. The numbering corresponds to the WHV7 sequence.27 qPCR was performed with the Applied Biosystems TaqMan Gene Expression Mastermix using each primer at a concentration of 600 nM and the TaqMan probe at a concentration of 250 nM. The reaction conditions were 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C, and 60 seconds at 60°C. To calculate cccDNA copy numbers, a 10-fold dilution series of NheI-linearized plasmid PUC-CMVWHV28 was used (range: 20-200,000 GE of WHV).