Evaluating SARS-CoV-2 PLpro and Fibrosis Genes

To measure the expression of SARS PLpro, TGF-β1, pro-fibrotic and pro-protein convertase genes in transfected cells or mouse lung tissues, total RNAs were extracted from transfected A549 cells with empty vector pcDNA3.1 or pSARS-PLpro 2 days post transfection using PureLink Micro-to-Midi Total RNA Purification System kit (Invitrogen). Relative mRNA levels were analyzed using two-step real time RT-PCR with SYBR Green I, as described in our prior reports11 (link)12 (link). Primer pairs of SARS PLpro, TGF-β1, pro-fibrotic and pro-protein convertase genes were listed in Table 1. Quantification of specific PCR products was performed using the ABI Prism 7900HT Sequence Detection System (PE Applied Biosystems). Relative changes in mRNA level of indicated genes were normalized relative to GAPDH mRNA.