Sybr green premix reagent

TRIzol^® reagent (Tiangen Biotech Co., Ltd., Beijing, China) was used to extract total RNA from cells. According to the manufacturer's protocols, 2 µg of RNA was used for cDNA synthesis using the First Strand cDNA Synthesis kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). RT-qPCR was performed using the SYBR Green Premix reagent (TakaraBio, Inc., Otsu, Japan) in an ABI 7500 Thermocycler (Thermo Fisher Scientific, Inc.). The PCR thermocycling conditions were as follows: Initial denaturation for 10 min at 95°C, 40 cycles of 95°C for 5 sec and 65°C for 31 sec, followed by 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and a final extension at 72°C for 10 min and finally held at 4°C. β-actin was used as the internal control for normalization. The primers used for RT-qPCR were listed in Table I.

Total RNA was extracted with Trizol reagent and quantified using SYBR Green Premix Reagent (Takara Bio, Shiga, Japan) and a TaqMan miRNA kit (Applied Biosystems) according to the manufacturer’s protocols. The endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 was used for normalization. The primers used are described in Table S8. Relative expression was quantified using the comparative threshold cycle (Ct) method.

Total RNA was extracted with Trizol reagent and quantified using real-time PCR using the SYBR Green Premix reagent (Takara Bio Inc., Shiga, Japan). The mRNA levels of Foxp3 were analyzed and normalized to Actb.

HBV DNA was detected in HepG2.2.15 cells treated with IFN-α or TDS (0.8, 1.6 and 3.2 mg/ml) using quantitative PCR. Total DNA was extracted from the cell supernatant using the TIANamp Virus DNA/RNA kit (Tiangen Biotech Co., Ltd., Beijing, China) and Wizard^® Genomic DNA Purification kit (Promega Corporation). The quantification of HBV DNA copies was performed using the SYBR green premix reagent (Takara Biotechnology Co., Ltd., Dalian, China). Total DNA (2 µg) was used as the template for each quantitative PCR assay. PCR was performed using an ABI 7900 real-time PCR detector (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 93°C for 2 min, 10 cycles at 93°C for 45 sec and 55°C for 60 sec, and 30 cycles at 93°C for 30 sec and 55°C for 45 sec. GAPDH served as a control gene. The primers utilized for HBV DNA fragment amplification were as follows: Forward primer, 5′-CCTCTTCATCCTGCTGCT-3′ and reverse primer, 5′-AACTGAAAGCCAAACAGTG-3′. GAPDH primers: Forward primer, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse primer, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. Assays were repeated in triplicate and mean quantification cycle values were used to calculate the levels of HBV-DNA using the 2^−ΔΔCq method (25 (link)). The inhibitory rate was calculated using the formula: Inhibitory rate (%)=[DNA copy (C) control-C sample]/C control ×100.