Lightcycler 4

Total RNA was isolated from leaves using the RNeasy Plant mini kit (Qiagen Inc., Tokyo, Japan) following the manufacturer’s instructions. Poly(A)⁺ RNA was purified from total RNA with the Poly (A) Purist MAG (Ambion Inc., Austin, TX, USA). Then, the purified poly(A)⁺ RNA was dissolved in the RNA storage solution. First-strand cDNA for quantitative RT-PCR was synthesized from poly(A) ⁺ RNA using a PrimeScript RT Master Mix (Takara Bio Inc., Shiga, Japan). Quantitative RT-PCR was performed in a mixture of 20 μL containing first-strand cDNA, SYBR Premix Ex Taq (Takara Bio Inc.), and 0.2 µmol of each primer combination (Table S1), using LightCycler 2.0 (Roche Applied Science, Mannheim, Germany). The thermal cycle profile was 1 cycle of 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. The cDNA quantities of the respective genes were calculated using LightCycler 4.0 software (Roche Applied Science, Penzberg, Germany) and were normalized with that of the glyceraldehyde 3-phosphate dehydrogenase gene [28 (link)]. Expression analyses were conducted three times.

To quantify miRNA expression, RNA was isolated from AT-hMSCs and DHCs using TRIzol reagent (Invitrogen) following the manufacturer's instructions. miRNAs expression was then measured using the miRNA detection service from Genolution Pharmaceutics; each sample was normalized to the expression of U6 miRNA.
The levels of HCV RNA in HCV-infected cells were also assessed by rqRT-PCR. An HCV-specific probe, 5′-FAM-CGC AGA CCA CTA TGG CTC TCC CG-BHQ-1-3′ (Metabion, Martinsried, Germany), was synthesized. Amplification was carried out using the LightCycler 480 system (Roche Applied Science, Basel, Switzerland) with a PCR mixture containing 5 pmol of forward primer (5′-ATG GCG TTA GTA TGA GTG TCG T-3′), 5 pmol of reverse primer (5′-YGG GTT KAT CCA AGA AAG GAC-3′), and 5 pmol of HCV-specific TaqMan probe. The thermal conditions were designed using the Roche Universal Probe Library's thermocycling conditions following the manufacturer's instructions. Human β-actin was used as a reference gene. All fluorescence data were analyzed using the LightCycler 4.0 software (Roche Applied Science), and C_t results were exported to Excel sheets. The comparative C_t method was used for relative quantification and normalization.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from 5 μg of total RNA using reverse transcriptase (Promega, Madison, WI) and random primers (Promega) and amplified using Lightcycler 480 Probes Master real-time PCR master mix (Roche Applied Science, Indianapolis, IN) in combination with Universal Probe Library (UPL) assays (Roche Applied Science). Assays were designed according to publicly available gene sequences (NCBI) using ProbeFinder UPL software (v.2.45) (Roche Applied Science). Each 20 μL PCR reaction comprised 0.4 μM target primers, 0.4 μM target UPL, 0.4 μM reference primers, 0.4 μM reference probe, and Roche real-time PCR master mix. The cycling conditions were as follows: preincubation at 95°C for 10 min, followed by 45 cycles at 95°C for 10 s, 55°C for 45 s, and 72°C for 1 s. Human β-actin and human glucose 6-phosphate dehydrogenase (G6PD) were used as reference genes. All fluorescence data were analyzed using LightCycler 4.0 software (Roche Applied Science), and C_t results were exported to Excel (Microsoft, Redmond, WA). Gene expression was quantified and normalized using the comparative C_t method.

RNA was extracted from biopsied hepatic tissues using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized according to manufacturer’s protocol, using 1 μg of total RNA using Superscript II (Invitrogen, Carlsbad, CA). Real-time polymerase-chain reaction (RT-PCR) was performed using a Light-Cycler 4.8 instrument (Roche Applied Science, Indianapolis, IN). cDNA was amplified using the ‘universal probe system’ on a Roche microplate with a final volume of 10 μL reaction mix containing 2.5 μL, 100-fold diluted cDNA, 7 μL LightCycler TaqMan Master Mix buffer (Roche Probes Master kit, Roche Applied Science, Indianapolis, IN), 1 μmol/L specific forward-reverse primers and 0.5 μL specific universal probes. Primers and universal probes are shown in Table 1. Quantification of 18S rRNA was used for sample normalization using TaqMan probes. RT-PCR was performed according to manufacturer’s protocol (Roche Applied science, Indianapolis, USA). The specificity of amplification was determined by melting curve analysis. The results were analyzed by relative quantification, ΔΔC method.

Total RNA was extracted from NCCIT cell line (four biological replicates) using TRIzol (Invitrogen, Carlsbad, CA, USA) and quantified in NanoDropTM Lite Spectophotometer (Cat. ND-LITE, Thermo ScientificTM, Waltham, MA, USA). Four hundred nanograms of cDNA were synthetized using Transcriptor High Fidelity cDNA Synthesis Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. The RT² Profiler PCR Array System Kit (Qiagen, Hilden, Germany) included 96 genes corresponding to cancer research molecular pathways and adequate controls, in quadruplicates. The expression levels were determined by real-time quantitative PCR in a LightCycler 48 platform (Roche Diagnostics, Risch-Rotkreuz, Switzerland) and ACTINβ, β2M, GAPDH, HPRT1, and RPLP0 were used as endogenous controls. The RT² profiler PCR array analysis was performed in Qiagen specific platform. The data analysis in web portal calculates fold change using ΔΔCt method. Genes with a logarithm fold change above 1.5 or below −1.5 were considered. Additionally, DNA genomic contamination, as well as first strand synthesis and real-time PCR efficiency were monitored in Qiagen platform for RT² profiler PCR array analysis. The lower limit detection was set at Ct ≥ 35.