Sensifast probe hi rox one step kit

Total RNA was extracted from RCFs using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s specifications. The RNA was quantified (NanoDrop ND-1000 spectrophotometer, Thermo Fisher Scientific). Quantitative real-time PCR was performed using equivalent amounts of RNA (varying 30–50 ng) with the SensiFAST Probe Hi-ROX One-Step kit (Bioline, Taunton, MA, USA) and TaqMan aptamers specific to rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Oc03823402_g1; Thermo Fisher Scientific) or αSMA (ACTA2, Oc03399251_m1; Thermo Fisher Scientific) in the total volume of 10 μL per reaction, as previously described [26 (link)]. The GAPDH expression served as a housekeeping control. Gene expression data were calculated as previously reported [12 (link)] and normalized to the expression of mRNA from cells in the absence of both TGF-β1 and andrographolide. The qPCR was performed from three independent experiments.

RNA was extracted with the ISOLATE RNA Mini Kit (Bioline) and quantified by real‐time PCR using SensiFAST™ Probe Hi‐ROX One‐Step Kit (Bioline) allowing reverse transcription and PCR amplification in a single step. The real‐time PCR was performed in a Roche LightCycler 96. The Renilla luciferase, β‐actin, and pBAC‐SARS‐CoV replicon qPCR primers are listed in Appendix Table S2. Experiments were performed in at least triplicates.

For siRNA-mediated knock-down of Plxdc2, 293T cells were plated one day prior to transfection in 6well plates. 10μl of a 5μM siRNA solution (set of 4x siCtrl or set of 4x siPlxdc2) purchased as ON-TARGETplus siRNAs from Dharmacon were transfected using 2μl fo DharmaFECT 1 transfection reagent (Dharmacon) according to manufacturer’s instructions in serum-free DMEM without antibiotics. 6h post transfection fresh DMEM supplemented with 20% FCS was added to achieve a final concentration of 10%FCS. 48h post transfection 293T siCtrl cells and 293T siPlxdc2 cells were harvested, cell numbers were determined and cells were plated at cells were plated at 50 000 cells/cm² for infection assays (see below). For determination of knock-down efficiency, cells were harvested in RNAzol 96h post transfection and RNA was isolated using the Direct-zol RNA Miniprep Plus Kit according to the manufacturer’s instructions. qPCR was performed on a StepOne Plus cycler (Thermo Fisher Scientific) in 20μl reactions with 40ng RNA/reaction using the SensiFAST Probe Hi-ROX One-Step Kit (Bioline) (cycling conditions: 10min 45°Cmin, 2min initial denaturation at 95°C, 40 cycles 95°C for 5s and 60°C for 20s). Primer-probe sets specific for Plxdc2 and GAPDH were used. Samples were analyzed in technical triplicates. See S2 Table for a complete list of primers.