Rnase free dnase

Total RNA was extracted from liver samples using TRI-Reagent (Sigma St. Louis, MO USA), followed by treatment with 1U of RNase-free DNase (Roche Basel, Switzerland). Reverse transcription was performed on 2 μg total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Foster City, CA USA), according to the manufacturer’s instructions. qPCR was performed on 50ng cDNA samples using the SYBR Green Real-Time PCR Kit (Applied Biosystems Foster City, CA USA), according to manufacturer’s specifications, with gene-specific primers (Table 1) in an ABI Step ONE Plus system (Applied Biosystems Foster City, CA USA). HPRT and β-Actin were used as an endogenous reference control. All reactions were performed in duplicates and relative gene expression values were determined using the 2.ddCt method with ABI Prism 7000 SDS (Applied Biosystems Foster City, CA USA). Results from the WT saline group were used as a reference group for the calculations.

Total RNA from HSC-3 cells was isolated using Trizol reagent (Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s recommendations. The cancer cell RNA was digested with RNase-free DNase (Roche; Basel, Switzerland) for 15 min at 37 °C and purified using the RNeasy kit (Qiagen; Hilden, Germany) according to the manufacturer’s protocol. cDNA was synthesized from 2 μg of total RNA by incubation at 37 °C for l h with avian myeloblastosis virus reverse transcriptase (GE Healthcare; Little Chalfont, United Kingdom) with random hexanucleotides according to the manufacturer’s instruction. Sequences of primers used to specifically amplify the genes of interest are shown in Table 1. Amplification was performed in a thermal cycler (Eppendorf; Hamburg, Germany). The polymerase chain reaction (PCR) products were separated in 1.0% agarose gels and visualized with ethidium bromide staining [11 (link)].

Total RNA was extracted by using Trizol reagent (Invitrogen) according to standard procedures [23 ]. After digestion with RNase-free DNase (Roche), total RNA was retro-transcribed with random primers using M-MLV reverse transcriptase (Promega) according to standard procedures [23 ]. qPCR was carried out using LightCycler 480 SYBR Green I Master and the LightCycler 480 System (Roche), according to manufacturer's instructions [23 ]. Primers used in RT-PCR and qPCR experiments are listed in Additional file 1: Supplementary Table 1.

The viral RNA was extracted from clarified culture supernatants using the NUCLISENS easyMAG system (bioMérieux, Marcy-l’Étoile, France) and digested with RNase-free DNase (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The viral RNA was detected by qRT-PCR using the primer pair RVA7-1F/Rota NVP3-R and the probe RVA7probe1, which were specific to RVA-NSP3, as described previously [44 (link)]. Genome copy equivalents (GCEs) were calculated by including a standard based on a dilution series of plasmid pT7-NSP3SA11. RT-PCRs were performed using the OneStep RT-PCR Kit (Qiagen) according to the manufacturer’s instructions. The primer sequences are listed in Table S1. RT-PCR products were analyzed on a 2% agarose gel. Prior to Sanger sequencing (Eurofins) or restriction digest analyses, amplicons were cleaned up using the Monarch DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA, USA).