Quantitative Real-Time Reverse-Transcription PCR (qRT-PCR) Analysis
This protocol is extracted from research article:
Inhibition of Human Coronaviruses by Antimalarial Peroxides
ACS Infect Dis, Mar 30, 2021; DOI: 10.1021/acsinfecdis.1c00053

Total cellular RNA was obtained from infected or noninfected cell cultures and isolated using the RNEasy Mini Kit (Qiagen, Germantown, MD), according to the manufacturer’s instructions. Secreted viral RNA was isolated from 50 μL of cell culture supernatants using the QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD). RNA was reverse transcribed to cDNA using the RevertAid first Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) and quantified by real-time RT-PCR using PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. All qRT-PCR assays included three sets of control wells: no reverse transcriptase, no cDNA template, or water only. The following primer pairs were used: OC43-F, 5′-GCTCAGGAAGGTCTGCTCC-3′; OC43-R, 5′-TCCTGCACTAGAGGCTCTGC-3′;17 NL63-F, 5′-AGGACCTTAAATTCAGACAACGTTCT-3′; NL63-R, 5′-GATTACGTTTGCGATTACCAAGACT-3′;17 GAPDH-F, 5′-TTGGTATCGTGGAAGGACTC-3′; and GAPDH-R, 5′-ACAGTCTTCTGGGTGGCAGT-3′.47 Real-time PCR was performed on a Bio-Rad CFX Connect system (Bio-Rad, Hercules, CA).

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