Super script 3 platinum one step rt qpcr

Frozen lungs and nasal turbinates fragments from the golden Syrian hamsters were weighted and homogenized with 1 mL of ice-cold DMEM (Gibco) supplemented with 1% penicillin/streptomycin (Thermo Fisher) in Lysing Matrix M 2 mL tubes (MP Biomedicals) using the FastPrep-24™ system (MP Biomedicals).
For viral titration, the tissue homogenate supernatants were titrated on Vero-E6 cells by classical plaque assays using semisolid overlays (Avicel, RC581-NFDR080I, DuPont) and expressed and PFU/100 mg of tissue⁶² .
For RNA isolation, the tissue homogenate supernatants were mixed with Trizol LS (Invitrogen) and the total RNA from nasal turbinates was extracted using the Direct-zol RNA MicroPrep Kit (Zymo Research) and from lungs with the (Zymo Research). The presence of SARS-CoV-2 RNA in these samples was evaluated by Superscript III Platinum One-Step RT-qPCR (Invitrogen) containing the nCoV_IP2 and the nCoV_IP2 probe (S.Table 2) according to the manufacturer’s protocol. Viral load quantification (RNA copy number/mg of tissue) was assessed by linear regression using a standard curve of RNA transcripts containing the RdRp sequence (ranging from 10⁷ to 10² copies).

Viral RNA was extracted from nasal turbinates and lung using QIAmp Viral extraction kit according to the manufacturer’s instructions. RT-qPCR assay was performed using the protocol established by the Institut Pasteur⁵³ . In brief, primers and probe targeting SARS-CoV-2 E gene were used following the Super Script III Platinum One-Step RT-qPCR (Invitrogen) protocol. Amplification was performed as followed: reverse transcription 55 °C 20 min, denaturation 95 °C 3 min, followed by 50x cycles of amplification at 95 °C 15 s, 58 °C 30 s, where data was acquired. Further analysis and Cq values were determined using the Bio-Rad CFX Maestro software (BioRad). Analysis was performed in quadruplicates.

Viral RNA was extracted using a QIAmp Viral extraction kit according to the manufacturer’s instructions. Reverse transcription-real-time polymerase chain reaction (RT-qPCR) assay was performed using the protocol established by the Institut Pasteur (20 (link)). In brief, primers and probe targeting SARS-CoV-2 RdRp gene were used following the Super Script III Platinum One-Step RT-qPCR (Invitrogen) protocol. Amplification was performed as followed: reverse transcription at 55°C for 20 min, denaturation at 95°C for 3 min, followed by 50× cycles of amplification at 95°C for 15 s and at 58°C for 30 s where data were acquired. Further analysis and Quantification Cycle (Cq) values were determined using the Bio-Rad CFX Maestro software (Bio-Rad).