Rneasy lipid tissue extraction kit

Two different RNA extraction methods were used based on tissue type. Total RNA was extracted using the RNeasy Lipid Tissue extraction kit (Qiagen) according to the manufacturer’s protocols. To each MagNA Lyser Green Beads vial (Roche), 1 mL QIAzol lysis reagent buffer (Thermo Scientific™) was added. Next, the tissues were homogenized for 30 s at 6,000 × g using a MagNA Lyser instrument (Roche). Finally, total RNA was eluted in 80 μL of nuclease-free water. By contrast, total RNA was extracted from the SOG and PG using the RNAqueous®-Micro Kit (Ambion) according to the manufacturer’s protocols. First, 100 µL of lysis solution was added to the tissues. Next, tissues were disrupted and homogenized using a Teflon micro pestle attached to a drill. Using either extraction method, a DNase treatment was performed upon elution of the extracted RNA. In both cases, the concentration of the resulting RNA extracts was determined by means of a Nanodrop spectrophotometer (Nanodrop ND-1000, Thermo Fisher Scientific, Inc.). RNA extracts were stored at −80°C.

Total RNA was isolated from the whole brain of uninfected and SARS-CoV-2 (Wu strain) infected K18-hACE2 mice (n = 4–9) using RNeasy Lipid Tissue extraction kit according to manufacturer’s instructions (QIAGEN, CA, USA). The total RNA was purified from genomic DNA contamination using Turbo DNase treatment (Ambion, NY, USA), then converted to cDNA using AffinityScript cDNA synthesis kit according to manufacturer’s instructions (Agilent Technologies, CA, USA). Commercially available gene specific TaqMan probes for Aif1 (Mm00479862_g1), Pycard (Mm00445747_g1), and Casp1 (Mm00438023_m1) were used to amplify target gene of interest (Applied Biosystems, MA, USA). Relative target gene expression to geometric mean of reference genes Gapdh (Mm99999915_g1) and Actb (Mm02619580_g1) was determined using this formula: 2^-∆CT where ∆CT = (Ct_{(Target gene)} – Ct_{(Gapdh and Actb)}). Final measures are presented as relative levels of gene expression in SARS-CoV-2 (Wu strain) infected K18-hACE2 mice compared with expression in uninfected K18-hACE2 mice. Probe set was tested over a serial cDNA concentration for amplification efficiency. Negative controls included no reverse transcription and water as a no template control. All samples were run in triplicate.