Hiscript qrt supermix gdna wiper

Whole thymi were dissected, and total cellular RNA was extracted by RNA Isolater Total RNA Extraction Reagent (Vazyme) following the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed using HiScript QRT SuperMix (+gDNA Wiper) (Vazyme) to generate cDNA. With ChamQ SYBR qPCR Master Mix (Vazyme), qPCR was performed on the Real-Time PCR detection system. All data were normalized to β-actin mRNA levels, and the 2^−△CT method was used to calculate the expression levels of target mRNAs. The primers used in the qPCR analysis are presented in Table S1.

Table S1. Primers for qPCR.

Total RNA from Marc-145 cells was extracted using total RNA kit I (Omega Bio-tek, Inc, Norcross, GA, USA) according to manufacturer’s instructions. RNA were converted to cDNA using HiScript^® Q RT SuperMix (+gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Two microliters of the RT reaction mixture were submitted to quantitative RT-PCR (qRT-PCR) using mViperin, PRRSV ORF7, IFN-α, or IFN regulatory factor-1 (IRF-1)-specific primers (Table 1), and SYBR Green Real-time PCR Master Mix (Vazyme), according to the manufacturer’s recommendations. The reaction procedure was 95°C for 5 min, followed by 40 cycles at 95°C for 5 s and 60°C for 31 s. Standard curve analysis was performed for relative quantification. The transcript levels of target genes were relatively quantified using the 2^-ΔΔCT method. The GAPDH gene served as an internal reference. The relative amount of target gene mRNA was normalized to that of GAPDH mRNA in the same sample.

Mimics and inhibitors of miR-497 were transfected into cells, and RNA and protein were extracted from transfected cells after 48 h. MiRNA expression levels were determined with the S-Poly (T) method (16). Total RNA (1 µg) was used for cDNA synthesis using a HiScript ^® QRT SuperMix (+ gDNA wiper) (Nanjing, China) kit for qPCR (R123-1, Vazyme, Nanjing, China). The primers were designed based on the sequences of bovine PPARG, ACLY, CD36, ELOVL6, HSL, and GAPDH in NCBI (Table 2), and real-time PCR results were analyzed using the 2^−ΔΔCt model.
SDS-PAGE was used to isolate proteins from cells, and they were transferred nitrocellulose membrane (Millipore, Billerica, MA, USA). Rabbit monoclonal (# 8579, Cell Signaling Technology, Shanghai, China) and monoclonal rabbit antibodies (# 8457, Cell Signaling Technology, Shanghai, China) were used, and image collection and analysis were conducted.