Gotaq probe qpcr and rt qpcr systems

The virus was incubated with a compound (1 µM) for 1 h and then added to monolayers of Vero E6 cells in 48-well plates (5 × 10⁵ cells/well) at an MOI of 0.01 for 1 h at 4 °C. The medium with the virus was removed, and cells were washed three times with medium, before lysis buffer addition. Total viral RNA was extracted using QIAamp Viral RNA (Qiagen, São Paulo, SP, Brazil), according to the manufacturer’s instructions. Quantitative RT-PCR was performed using GoTaq^® Probe qPCR and RT-qPCR Systems (Promega, Madison, Wisconsin, EUA) in a StepOne™ Real-Time PCR System (Thermo Fisher, Waltham, MA, USA). Amplifications were performed as 25 µL reactions containing 1× reaction mix buffer, 50 µM of each primer, 10 µM of the probe, and 5 µL of RNA template. Primers, probes, and cycling conditions followed the recommendations of the Centers for Disease Control and Prevention (CDC) protocol for the detection of the SARS-CoV-2 [63 ]. In addition, a virus quantification standard curve was included [64 (link)].

For detection of SLC19A1, HepaRG were treated with MTX for 3 days; for ACTA2 and DDIT3 detection, mono- and co-cultures were exposed to MTX or TGF-β1 for 7 days. mRNA was isolated following standard TRIzol extraction procedure with Glycogen (ThermoFisherScientific, Reinach, Switzerland, LT-02241) from cells lysed using QIAzol Lysis Reagent (Qiagen, Basel, Switzerland, 79306). Reverse transcription was carried out using M-MLV Reverse transcriptase (Promega, Dübendorf, Switzerland, M1705) and oligo dT (Qiagen, Basel, Switzerland, 79237). Real-time PCR was performed using either FastStart TaqMan^® Probe Master (Sigma, Taufkirchen, Germany, 4673417001; for SLC19A1) or GoTaq^® Probe qPCR and RT-qPCR Systems (Promega, Dübendorf, Switzerland, A6102; for DDIT3 and ACTA2) and TaqMan probes of selected genes (see Table 2). The q-RT-PCR Program used: 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The Ct values were generated using the Corbett Rotorgene Analysis Software 6000 (SLC19A1) or the LightCycler^® 480 Systems (DDIT3 and ACTA2) and processed on GraphPad Prism using B2M as the internal standard for normalization. Data are expressed as fold change.