Stat 60 rna extraction reagent

Lungs RNA was extracted using RNA-STAT 60 extraction reagent (Tel-Test, Inc.) plus chloroform, precipitated, and re-suspended in RNAse-free water. Control RNA was isolated from SARS-CoV-2 viral stocks following the same procedure and quantified by OD260. These control stocks were serially diluted and OD260_nm values measured to generate a standard curve. RT-qPCR of the lung RNA was carried out using the following primers: 2019-nCoV_N1-F: 5'-GAC CCC AAA ATC AGC GAA AT-3'; 2019-nCoV_N1-R: 5'-TCT GGT TAC TGC CAG TTG AAT CTG-3'; and probe 2019-nCoV_N1-P: 5'-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3' (Integrated DNA Technologies) which were designed to bind to and amplify a conserved region of SARS-CoV-2 Nucleocapsid (N) RNA. Amplification was performed with an Applied Biosystems 7500 Sequence detector using the following program: 48°C for 30 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 seconds, and 1 minat 55°C Reactions were carried out using a TaqMan RT-PCR kit (Meridian Bioscience, cat#BIO -78005) in 50 µL volume containing 5 µL of template, 2 µM of each primer and 2 µM of each probe. The number of viral RNA copies per mL was calculated by extrapolation from the standard curve, and values were then converted to the number of viral RNA copies per gram of lung tissue.

RNA from lungs of mice #M2–M4 and #F11-F13 was extracted using RNA-STAT 60 extraction reagent (Tel-Test, Inc., Friendswood, TX, USA) + chloroform, precipitated and resuspended in RNAse-free water. Control RNA was isolated from SARS-CoV-2 viral stocks following the same procedure and quantified by OD260. These control stocks were serially diluted and OD260 values measured to generate a standard curve. RT-qPCR of the lung RNA was carried out using the following primers : 2019-nCoV_N1-F :5’-GAC CCC AAA ATC AGC GAA AT-3’; 2019-nCoV_N1-R: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’; and probe 2019-nCoV_N1-P: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1–3’ (Integrated DNA Technologies, Coralville, IA, USA) which were designed to bind to and amplify a conserved region of SARS-CoV-2 Nucleocapsid (N) RNA. Amplification was performed with an Applied Biosystems 7500 Sequence detector using the following program: 48°C for 30 minutes, 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, and 1 minute at 55°C. Reactions were carried out using a TaqMan RT-PCR kit (Meridian Bioscience, Memphis, TN, USA) in 50 μL volume containing 5 μL of template, 2 μM of each primer and 2μM of each probe. The number of viral RNA copies per mL was calculated by extrapolation from the standard curve, and values were then converted to the number of viral RNA copies per gram of lung tissue.