4.7. Quantitative Real-Time PCR

A quarter of the total DNA extracted from intracellular HBV particles was used for quantification. For quantification of HBV RNA transcription, 2 μg of total RNA was synthesized into cDNA using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Each reaction mixture (15 μL) contained 2 μL of DNA (10- or 20-fold diluted) or cDNA (4-fold diluted), 0.4 μM of each primer, and 7.5 μL of SYBR green master mix (Applied Biosystem). Amplification conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min in QuantStudio 3 Real-Time PCR System (Applied Biosystem). Relative replication levels were calculated by the comparative 2^−∆∆CT method [11,13]. The levels of HBV RNA were normalized to GAPDH gene (NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NC_000012.12","term_id":"568815586"}}NC_000012.12). PCR primers were as follows: HBV DNA (or RNA); forward 5′-CTC GTG GTG GAC TTC TCT C-3′, reverse 5′-CTG CAG GAT GAA GAG GAA-3′ GAPDH; forward 5′-ATC ATC CCT GCC TCT ACT GG -3′, reverse 5′-TGG GTG TCG CTG TTG AAG TC-3′.