SARS-CoV-2 N Gene Quantification in Mouse Tissues

Mouse tissues (lung, brain and heart) were harvested on 3 dpi and were homogenized in sterile PBS (pH7.2) (10%, v/w) using a Fisherbrand^™ Bead Mill 24 Homogenizer (FisherScientific) followed by total RNA extraction using RNeasy Plus Mini Kit (Qiagen #74136). Extracted RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific #4368813). SARS-CoV-2 nucleocapsid (N) gene in individual mouse organs was determined using QuantiNova SYBR Green PCR kit (Qiagen #208052) in combination of 500 nM of 2019-nCoV RUO Kit (Integrated DNA Technologies #10006713). The cycling program was performed in Stratagene MX3000p qPCR system (Agilent) as follows: 95 °C for 120 s, 95 °C for 5 s (50 cycles) and 60 °C for 18 s^{29 (link)}. Threshold cycle (Ct) values were determined using MxPro qPCR software (Agilent). A standard curve was constructed using serially diluted pCC1-CoV2-F7 plasmid expressing SARS-CoV-2 N ^{36 (link)} and was used to interpolate the number of SARS-CoV-2 N gene copies in mouse tissues. A value of 1 was assigned if gene copies were below the detection limits.