Eagletaq universal master mix rox

Total RNA was extracted by Trizol reagent (Life Technologies Lofer, Salzburg, Austria), according to the manufacturer's instructions. The total RNA concentration was determined spectrophotometrically in RNAase-free water, and 1 μg aliquots of total RNA were reverse transcribed into cDNA using ProtoScript First Strand cDNA Syntesis Kit (New England BioLabs, Ipswich, MA). RT-PCR was carried out on ABI 7300HT sequence detection system (ABI), in a total volume of 20 μl containing EagleTaq Universal Master Mix (ROX) (Roche, Penzberg, Germany), DEPC water, 5 μl of cDNA. The following Prime Time qPCR Assay: TRPV1 qRnoCIP0024978 and TRPV4 qRnoCIP0027857 were purchased from Biorad (Biorad Laboratories; Hercules, California, USA). Quadruplicate samples were run for each gene. The reference gene GAPDH qRnoCIP0050838 were used as an internal control to normalize the expression of target genes. Relative expression levels were calculated for each sample after normalization against reference gene, using the ΔΔCt method for comparing relative fold expression differences, as previously described [48 (link)].

HBV DNA was extracted using SMITEST EX-R&D Kits (Medical & Biological Laboratories Co., Ltd., Tokyo, Japan) from the culture supernatants of Hep38.7-Tet cells and HBV-infected HepG2-hNTCP-C4 cells and PXB hepatocytes. HBV DNA was quantified by qPCR using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The primers and probes for HBV DNA, which were located in the S gene, were used as previously reported [45 (link),46 (link)], and they consisted of the forward primer 5′-CAC ATC AGG ATT CCT AGG ACC-3′, the reverse primer 5′-AGG TTG GTG AGT GAT TGG AG-3′, and the TaqMan probe 5′-(FAM)-CAG AGT CTA GAC TCG TGG TGG ACT TC-[6-carboxytetramethylrhodamine (TAMRA)]-3′. qPCR was performed by DNA polymerase activation at 95 °C for 10 min, followed by 45 cycles of denaturation (95 °C for 15 s) and annealing and elongation reaction (65 °C for 1 min) using EagleTaq Universal Master Mix (ROX) (F. Hoffmann-La Roche, Ltd., Basel, Switzerland).