Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro

Nicola Clementi; Elena Criscuolo; Roberta Antonia Diotti; Roberto Ferrarese; Matteo Castelli; Roberto Burioni; Massimo Clementi; Nicasio Mancini

Viral RNA extraction and real-time RT-PCR. Viral RNA was purified from 140 mL of cell culture supernatant using the QIAamp Viral RNA Mini Kit (QIAGEN), following the manufacturer's instructions. Subsequently, the purified RNA was used to perform the synthesis of first-strand cDNA, using the SuperScript™ First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific), following the manufacturer's instruction. Real-time PCR, using SYBR® Green dyebased PCR amplification and detection method, was performed in order to detect the cDNA. We used the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) the forward primer N2F: TTA CAA ACA TTG GCC GCA AA, the reverse primer N2R: GCG CGA CAT TCC GAA GAA, and the following PCR conditions: 95°C for 2 min, 45 cycles of 95°C for 20 sec, annealing at 55°C for 20 sec and elongation at 72°C for 30 sec, followed by a final elongation at 72°C for 10 min 7 . RT-PCR was performed using the ABI-PRISM 7900HT Fast Real-Time instruments (Applied Biosystems) by using optical-grade 96-well plates. Samples were run in duplicate in a total volume of 20 μL.