The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism

Wang, Yi; Liu, Li

Reverse transcription-PCR (RT-PCR) and qRT-PCR. Total RNAs were extracted from the cultured cells with TRIzol (Invitrogen, Carlsbad, CA, USA). The purified total RNAs were treated with DNase I (Qiagen, Düsseldorf, Germany). All primers used in the RT-PCRs are listed in Table 1 . The RT-PCR was carried out with the PrimeScript one-step RT-PCR kit (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions. The RT-PCR was carried out in a DNA Thermal Cycler (Applied Biosystems, Carlsbad, CA) under the following conditions: 50°C for 35 min for reverse transcription and 94°C for 5 min for denaturation. The PCR conditions were 94°C for 30 s, 50°C for 30 s, and 72°C for 50 s, repeated for 20 to 30 cycles; the reaction was extended at 72°C for 10 min before the reaction product was stored at 4°C. One-step real-time quantitative RT-PCR (qRT-PCR) (TaKaRa Biotechnology, Dalian, China) was also performed to monitor the targeted gene expression. The primers used in qRT-PCR were also listed in Table 1 . Real-time qRT-PCR was carried out with the CFX real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: 42°C for 5 min and 95°C for 10 s, and then 95°C for 5 s and 60°C for 10 s, repeated for 40 cycles. The dissociation of the reaction products was conducted from 55°C to 95°C as the temperature rose at 0.2°C per 10 s.