Sybr green real time pcr master mixes

HEK293T cells were transfected with pCAGGS-Flag-APPV-N^pro or pCAGGS-Flag-CSFV-N^pro for 24 h, followed by stimulation with Poly (I:C) for 12 h. Total RNA was extracted using total RNA Kit I (Takara, China) following the manufacturer’s protocol. The mRNA level of IFN-β was detected by RT-qPCR with SYBR Green Real-Time PCR Master Mixes (Takara). The β-actin was used as the reference gene. All data were showed as relative fold change by the threshold cycle (∆∆Ct) method.

Total RNA was extracted from HUVECs using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNAs using random primers (Sangon, Shanghai, China) and M-MLV reverse transcriptase (Promega, Madison, WI, USA), as per the manufacturer’s guidelines. Quantitative PCR (qPCR) was then performed using SYBR Green Real-Time PCR Master Mixes (TaKaRa, Shiga, Japan) based on the manufacturer’s protocol. The primer sequences used are listed in Table S1.

Total RNA was extracted using an RNAiso Plus kit from Takara (Dalian, China). Total miRNAs were extracted and purified using a TaqMan® miRNA ABC Purification Kit from ThermoFisher Scientific (Carlsbad, CA, USA) (cel-miR-39-3p was added as spike-in as previously reported [20 (link)]). After cDNA of lncRNA-MEG3 was amplified with M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA), qRT-PCR was carried out using SYBR Green Real-Time PCR Master Mixes (Takara, Dalian, China) (using GAPDH as internal control); to detect the levels of miR-223-3p in serum, the cDNAs were amplified and applied for qRT-PCR analysis using a mirVana™ qRT-PCR miRNA Detection Kit from Invitrogen (Carlsbad, CA, USA). The primers were designed using the OligoArchitect™ Online service (https://www.sigmaaldrich.com/china-mainland/zh/technical-documents/articles/biology/oligoarchitect-online.html) and miRprimer (https://sourceforge.net/projects/mirprimer/), respectively (Table 2) and synthesized by GenePharma (Shanghai, China).