2.7. Extraction and Reverse Transcription (RT) of HBV pgRNA

HBV RNA was extracted from 50 μL of serum using the Total RNA Extraction Miniprep System Kit according to the manufacturer’s instructions (Viogene, Taipei, Taiwan) and treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA). The isolated HBV RNA was reverse-transcribed using RevertAid reverse transcriptase (Thermo Fisher Scientific) with HBV-specific RT primers for HBV pgRNA. Before commencing RT, 20 μL of RNA, 1 μL of 10 μM primer, and 1 μL of 10 mM dNTPs (Thermo Fisher Scientific) were mixed, incubated at 70 °C for 5 min, and then placed immediately on ice for 1 min. RT was initiated by adding an RT reaction mix to a final volume of 35 μL at a final concentration of 1× RT buffer, 1 μL of RNAse inhibitor (Life Technologies, Carlsbad, CA, USA), and 1 μL of RevertAid reverse transcriptase (Thermo Fisher Scientific). The cycling conditions were as follows: 42 °C for 60 min followed by 75 °C for 5 min. Complementary DNA (cDNA) samples were stored at 4 °C before proceeding to quantitative real-time polymerase chain reaction (qPCR).