Takara primescript rt master mix

Total RNA was extracted from cells using TRIzol reagent (15596026; Thermo Fisher Scientific), and 1 μg of total RNA was reverse transcribed into cDNA using the TaKaRa PrimeScript RT master mix (TaKaRa Bio, Otsu, Japan). Five microliter of cDNA was used for performing quantitative PCR. Quantitative PCR was performed on a Roche LightCycler 480 (Roche Diagnostics, Palo Alto, CA, USA) using SYBR Premix Ex Taq II (Tli RNaseH Plus; TaKaRa Bio). The cycling program involved heating at 50 °C for 2 min, 95 °C for 10 min, followed by 35 cycles at 95 °C for 15 s 60 °C for 1 min. β-actin was used as the internal control. The following primers were used: AMIGO2-F: 5′-CCCCTGCAAGTGTAAAACCA-3′, AMIGO2-R: 5′-AGGGGTTCCAAAAACACCAC-3′; β-actin-F: 5′-AGAGGGAAATCGTGCGTGAC-3′, β-actin-R: 5′-CAATAGTGACCTGGCCGT-3′.

For mRNA quantification, RNA was extracted from cultured cells by the QuickGene RNA cultured cell kit S (RC-S) and a 50 μL volume of RNA was obtained using QuickGene-810 equipment, according to the manufacturer’s instructions (KURABO, Osaka, Japan). In a 20 μL reaction, total RNA (0.5 μg) was converted to cDNA using the reverse transcription Takara PrimeScript RT Master Mix (TaKaRa Bio, Shiga, Japan) and diluted using qPCR Takara EASY Dilution. The Brilliant III SYBR® MM with ROX PCR master mix (Agilent Technologies, CA) was used to prepare the diluted cDNA samples for quantification by the Agilent Mx3000P QPCR system. The following primer sets were used for mRNA quantification: TARDBP, 5′-CCCCAGATATTGCCAATATC-3′ and 5′-AAGTTTCCAATATGCTCAATTAAG-3′; Precore, 5′-GGTCTGTTCACCAGCACCAT-3′ and 5′-GGAAAGAAGTCAGAAGGCAA-3′; Core, 5′-CCGGAAAGCTTGAGCTCTTCTT-3′ and 5′-CACAGAATAGCTTGCCTGAGTG-3′; APOA2 5′-CAACTGTGCTACTCCTCACCAT-3′ and 5′-TGGAAGTACTGAGAAACCAGG C-3′; Total HBV RNA, 5′-GAGTGCTGTATGGTGAGGTG-3′ and 5′-TTTGGGGCATGGACATTGAC-3′. Quantification of HBV mRNAs (precore and pregenomic) was performed as we described recently^{62 (link)}. Gene expression was normalized to that of GAPDH. Relative mRNA levels represent the level of the gene divided by the level of GAPDH mRNA.

RNA was reverse transcribed into cDNA using Takara PrimeScript RT master mix (RR036A; Takara Biotechnology Co., Ltd., Dalian China). RT-qPCR was performed using an ABI StepOne Plus Real-Time PCR system (Thermo Fisher Scientific, Inc.) and SYBR Premix Ex Taq II master mix (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The reaction system (10 µl) consisted of cDNA (1 µl), forward primers (10 µM; 0.2 µl), reverse primers (10 µM; 0.2 µl), ROX reference dye (0.2 µl), RNase-free water (3.4 µl), and SYBR-Green mixture (5 µl). The thermocycling conditions were as follows: Initial denaturation, 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Rat actin was used as a housekeeping gene. The relative expression of genes was calculated using the 2^−∆∆Ct method [5 (link)].

RAW264. 7 cells of each group were seeded with a density of 5×10⁴ cells per well in a 24-well plate and incubated at 37°C for 4 days. HGF-1 cells of each group were seeded with a density of 5×10⁴ cells per well in a 24-well plate and incubated at 37°C for 7 days. Total RNA was extracted from cells using the TaKaRa MiniBEST Universal RNA Extraction KIT (TaKaRa, Japan) following the manufacturer’s protocol. An absorbance ratio of 260:280 was used to evaluate RNA purity, and its concentration was calculated by the absorbance at 260 nm. Total RNA was immediately reverse-transcribed into first-strand cDNA by the TaKaRa PrimeScript™RT Master Mix (TaKaRa, Japan). The obtained cDNA was stored at -20°C and used as soon as possible. As a qRT-PCR reaction template, the cDNA was used SYBR Premix Ex Taq II (TaKaRa, Japan) to amplify target genes on a Q7 system (QuantStudio 7, Applied Biosystems, USA). The reaction was carried out in a program of 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute and 95°C for 15 seconds, 60°C for 1 minute. 2^−ΔΔCt analysis method was used for calculating the relative expression of target genes. The target genes expressions were normalized to housekeeping gene β-actin. The primer sequences for qRT-PCR analysis were purchased from BioTNT (Shanghai, China) and were shown in Tables S1, S2.