Quantitative RT-PCR Analysis of Inflammatory and Viral Response Genes

NRCM (3.0 × 10⁴ cells/well) were incubated at least 24 h before treatment. Total RNA was isolated with a TRI reagent (Sigma, Burligton, MA, USA) from NRCM treated by S protein for 3 h. cDNA was synthesized with a ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed with ABI prism 7500 Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) and KAPA SYBR FAST qPCR kit (Roche, Basel, Switzerland). TNF-α primers were forward 5′- AAATGGGCTCCCTCTCATCAGTTC-3′ and reverse 5′- TCTGCTTGGTGGTTTGCTACGAC-3′ [61 (link)]. IL-1β primers were forward 5′-CACCTCTCAAGCAGAGCACAG-3′ and reverse 5′-GGGTTCCATGGTGAAGTCAA-3′ [61 (link)]. IL-6 primers were forward 5′-TCCTACCCCAACTTCCAATGCTC-3′ and reverse 5′-TTGGATGGTCTTGGTCCTTAGCC-3′ [61 (link)]. ACE2 primers were forward 5′- GCAGATGGCTACAACTATAACCG-3′ and reverse 5′-C CCTCCTCACATAGGCATGAAGA-3′. A total of 18 s rRNA amplification were forward 5′-ATTAATCAAGAACGAAAGTCGCAGGT-3′ and reverse 5′-TTTAAGTTTCAGCTTTGCAACCATACT-3′. Data were normalized with 18 s rRNA.