Serial dilutions of a plasmid containing a monomeric HBV insert selleck Ixazomib (Alfa Wasserman, Milan, Italy) were used as quantification standards. To perform quantification of cccDNA, aliquots of liver DNA extracts were treated for 1 h at 37��C with 10 U of plasmid-safe DNase (Epicentre, Madison, WI). Real-time PCR experiments were then performed using a 75-��l reaction volume containing 20 ng of DNA, 3 mM MgCl2, 0.5 ��M (each) forward and reverse primers, 0.2 ��M 3��-FL-labeled probe, and 0.4 ��M 5��-R640-labeled probe. Amplification was performed under the following conditions: 95��C for 10 min and then 60 cycles of 95��C for 30 s, 58��C for 10 s, 63��C for 30 s, and 72��C for 20 s.
The efficacy of DNase treatment in the elimination of open circular and single-stranded forms of HBV DNA prior to PCR was confirmed by abrogation of the PCR amplification of HBV DNA extracted from serum viral particles by use of nonselective HBV-specific oligonucleotide primers targeting the HBs open reading frame. To normalize the number of viral genomes present in each liver sample, the number of haploid genomes was evaluated by using a ��-globin gene kit (Roche DNA control kit; Roche Diagnostics). To evaluate intrahepatic concentrations of relaxed circular replicative DNA (rcDNA), cccDNA amounts were subtracted from total intracellular HBV DNA values. Molecular analyses of HDV RNA from serum samples. HDV RNA was extracted from 200 ��l of serum by use of TRIzol reagent (Invitrogen, Paisley, Scotland) as recommended by the manufacturer and then was dissolved in 50 ��l diethyl pyrocarbonate (DEPC)-treated water.
Five microliters of extracted RNA was used as a template for first-strand cDNA synthesis by use of a Super Script reverse transcriptase kit (Invitrogen) and oligo(dT) primers. HDV genotype determination was performed through reverse transcription-PCR (RT-PCR) amplification and sequencing analysis of the R0 region of the HDV genome, which covers the 3�� end of the HD gene (nucleotides 885 to 1285 [numbering according to reference 33]), as previously described by Radjef et al. (17). Primers and probes for HDV real-time PCR assays were designed by taking into account the genetic variability of HDV.
The sequences and nucleotide positions of the primers and probes were as follows: DELTAF, 5��-AAGGGGGACTCCGGGACTC-3�� (nt 1063 to 1081); DELTAR, 5��-CCTCAGCAAGGAGGAAGAAGA-3�� (nt 1236 to 1216); DELTA/FL (FRET hybridization probe), 5��-CAGACTGGGGACGAAGCCGCCCC-FL-3�� Brefeldin_A (nt 1086 to 1108); and DELTA/LC (FRET hybridization probe), 5��-LC-R640-CGCTCCCCTCGATCCACCTTCGAGG-PH-3�� (nt 1113 to 1137). Real-time PCR by use of the ��utility channel�� of a Cobas TaqMan 48 instrument was performed under the following conditions: 95��C for 10 min and then 60 cycles of 95��C for 30 s, 57��C for 20 s, and 72��C for 20 s. The plasmid pCRII-delta-R0, containing one copy of the R0 region of the HDV genome (nt 853 to 1308), was used as a standard for HDV cDNA quantification.