Rneasy extraction kit

Total RNA was isolated from breast (MDA-MB-231 and MCF-7), colon (SW620, SW480, and Colo205), prostate (LNCaP), and ovarian (OV17-1) tumor cell lines using the RNeasy extraction kit (Quiagen, Valencia, CA) and reverse-transcribed into cDNA with the Advantage RT-PCR kit (Clontech, Mountain View, CA). cDNA (100 ng) was evaluated by RT-PCR using primers for Ang1 (Hs00375822_m1), Ang2 (Hs01048042_m1), Tie2 (Hs00945146_m1), and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (4326317E) (Applied Biosystems, Carlsbad, CA). Expression of each gene of interest was normalized to GAPDH. The assay was performed using the 7300 RT-4CR System (Applied Biosystems).

The mRNA was extracted from the cells by a silicate gel technique using the Qiagen RNeasy extraction kit (Qiagen Inc., Hilden, Germany), which includes a DNAse digestion step. The amount of extracted mRNA was measured by UV spectrophotometry at 260 nm (Eppendorf AG, Hamburg, Germany), and contamination with proteins was determined according to the 260/280 ratio. An equal amount of RNA (1 μg total RNA in 40 μL total volume) was reverse-transcribed to cDNA and amplified by PCR with the iScript™ cDNA Synthesis Kit (Bio-Rad laboratories, Hercules, CA, USA), following the manufacturer’s instructions.

For reverse transcriptase PCR (RT-PCR), total RNA was extracted from Calu-3 cell culture infected with P. aeruginosa PAO1 wild type or ∆pqsA using a Qiagen RNeasy extraction kit (Qiagen). cDNA synthesis was performed from 1 μg of total purified RNA by using random hexamer primers and GoScript reverse transcriptase (Promega). A total of 50 ng of resulting cDNA was PCR-amplified using Expand High Fidelity PCR System (ROCHE) and primers pqsERT For and pqsERT Rev (for pqsE), pqsART For and pqsART Rev (for pqsA), oprLRT For and oprLRT Rev (for oprL), mexGRT For and mexGRT Rev (for mexG), and lecART For and lecART Rev (for lecA) (Table S3). After 5 min of denaturation at 95°C, the following reaction cycle was used for 35 cycles: 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The PCR products were analyzed on a 2% (w/v) agarose gel and stained with Tracklt Cyan/Orange (Invitrogen).

Lungs were collected 4 dpi and homogenized in 1.0 ml of DMEM, clarified by centrifugation (1,000 g for 5 min) and stored at −80 °C. Nasal washes were clarified by centrifugation (2,000 g for 10 min) and the supernatant was stored at −80 °C. To quantify viral load in lung tissue homogenates and nasal washes, RNA was extracted from 100 µl samples using E.Z.N.A. Total RNA Kit I (Omega) and eluted with 50 µl of water. Four microlitres of RNA was used for real-time quantitative PCR with reverse transcription to detect and quantify the N gene of SARS-CoV-2 using TaqMan RNA-to-CT 1-Step Kit (Thermo Fisher Scientific) as described previously^{60 (link)}. The virus titres in the nasal turbinates and lungs were determined by plaque assay on Vero E6 cells expressing human TMRPSS2 as previously published⁶¹ . RNA was extracted from clarified nasal washes using the Qiagen RNeasy extraction kit (Qiagen) following the manufacturer’s instructions. Samples were purified on the included columns and eluted in 50 µl of nuclease-free water. PCR was conducted using 4× TaqMan Fast Virus Master Mix (Thermo Fisher) and an N-gene primer/probe set.