Total RNA from HBV-infected cells was isolated with Trizol reagent (Invitrogen). About 400 ng total RNA was reverse transcribed into cDNA with PrimeScript RT kit (Takara) in a 10 μl reaction. cDNA derived from 20 ng total RNA was used as template for real-time PCR amplification. In a separate realtime PCR reaction, 20 ng of total RNA was directly used as template to assess the possible HBV viral DNA contamination in the RNA preparation. Primers (HBV2270F: 5′-GAGTGTGGATTCGCACTCC-3′) and (HBV2392R: 5′-GAGGCGAGGGAGTTCTTCT-3′) were used for HBV 3.5 kb transcripts; (HBV1805F: 5′-TCACCAGCACCATGCAAC-3′) and (HBV1896R: 5′-AAGCCACCCAAGGCACAG-3′) were for total HBV-specific transcripts. Amplification of 123-bp fragment for 3.5 kb transcripts and 92-bp product for total HBV-specific transcripts were both conducted by denaturation at 95°C for 30 s, followed by 40 cycles of 95°C denaturation for 3 s, and 60°C annealing/elongation for 30 s. Real-time PCR was performed using SYBR Premix Ex Taq kit on an ABI Fast 7500 real-time system instrument. Real-time PCR using either set of the primers generated highly specific amplification product. HBV RNA copy numbers were deduced from a standard curve generated from known nucleic acid quantities. Then the HBV RNA copy number per nanogram RNA in the infected cell cultures was calculated by subtracting the background amplification noise derived from the viral DNA contamination in the RNA preparation from that cDNA amplification. The signal-to-noise ratio for HBV total transcripts is usually ≥50, and for 3.5 kb transcripts ≥20. The real-time PCR detection limits for total HBV-specific transcripts and 3.5 kb transcripts are ∼0.5 and ∼3.5 copies per nanogram cellular total RNA, respectively.