Qscript xlt one step rt qpcr toughmix kit

Lung sections were homogenized using the Tissue Lizer II (Qiagen, Gaithersburg, MD) by adding 1 mL of PBS to each tube containing the sample and a Tungsten carbide 3 mm bead (Qiagen, Gaithersburg, MD) and then homogenizing for 10 minutes at 30 Hz. RNA from tissue and nasal swab samples was then extracted using the MagMax-96 AI/ND viral RNA isolation kit. Tissue samples were then normalized to 1 μg total RNA in 20 μL of nuclease-free water while RNA extracted from nasal swabs was used directly in the RT-qPCR reaction. The one-step RT-qPCR was performed using the Quantabio qScript XLT one-Step RT-qPCR ToughMix kit as described above and FLUAV TCID₅₀ equivalent per μg of total RNA titers (TCID₅₀eq/μg total RNA) in tissue sections was calculated according to a standard curve of an exact match of virus stock of known titer.

RNAs were extracted from the different tissue homogenates using the MagMax-96 AI/ND viral RNA isolation kit (ThermoFisher Scientific, Waltham, MA) following manufacturer’s protocol. A one-step real time quantitative PCR (RT-qPCR) based on the Nucleoprotein gene segment was used as surrogate of viral load and it was employed using the primers 2019-nCov_N2-F (5′-TTACAAACATTGGCCGCAAA-3′) and 2019-nCov_N2-R (5′-GCGCGACATTCCGAAGAA-3′). A probe with FAM as a reporter and TAMRA as a quencher was used (5′-FAM-ACAATTTGCCCCCAGCGCTTCAG-TAMRA-3′). The RT-qPCR was performed in a QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA) using a Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly, MA) in a final reaction volume of 20 μl. Each reaction mixture contained 1 × master mix, 0.5 μM forward and reverse primers, 0.3 μM probe, and 5 μl of RNA. The qPCR cycling conditions were 50 °C, 20 min; 95 °C, 1 min, 40 cycles at 95 °C, 1 min; 60 °C, 1 min; and 72 °C 1 s; with a final cooling step at 4 °C. A standard curve was generated using tenfold serial dilutions of a SARS-CoV-2 virus stock of known titer to correlate RT-qPCR crossing point (Cp) values with the viral load from each tissue. Viral loads were calculated as Log₁₀ TCID50 equivalents/per gram of tissue.