Itaq master sybr green super mix

The total RNA of lung tissues was extracted using a FastPure^® Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s instructions. First-strand cDNA was synthesized using HiScript^® III SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China), and stored at −20 °C. A real-time PCR reaction system was prepared using the iTaq Master SYBR Green Super Mix (Bio-Rad Laboratories Inc., Hercules, CA, USA) and carried out with a CFX96 Thermal Cycler (Bio-Rad Laboratories Inc.). The expressions of genes were calculated using the 2^−ΔΔCT method, and normalized to that of GAPDH. Table 1 shows the primer sequences.

Total RNA was extracted from lung tissues using the FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China). First-strand cDNA was synthesized from total RNA using HiScript^® III SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed using iTaq Master SYBR Green Super Mix (Bio-Rad Laboratories Inc., Hercules, CA, United States) and a CFX96 Thermal Cycler (Bio-Rad Laboratories Inc.). The relative expression of genes was normalized to that of GAPDH and calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Table 1 shows the primer sequences.

The expressions of MxA, TLR7, MyD88, TRAF6, and TNF-α were determined using quantitative real-time PCR (qPCR). Changes in viral nuclear proteins (NP) was used to characterize virus loading. After sacrifice, the lung section was collected and homogenized with 0.01 M phosphate-buffered saline (pH 7.20) at 65 HZ for 10 min. RNA was extracted from the lung tissues using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instruction. The qPCR was performed using iTaq Master SYBR Green Super Mix (Bio-Rad, Hercules, CA, USA) in a RT-PCR system (Thermal cycler CFX96, Bio-Rad, Hercules, CA, USA). The relative expression of genes was normalized to that of GAPDH and calculated according to the 2^−ΔΔCT approach. Primers used are shown in Table 1.