QuantiNova Pathogen +IC Kit (500)

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Publication protocol

Quantitative (q) PCR: The total RNAs from the challenged IFNAR−/− mice organs (brain, spleen, and liver) were extracted using TRI-REAGENT (BioShop Inc, Burlington, Ontario, Canada), in accordance with the manufacturer’s instruction. Briefly, the tissue samples were homogenized in a TRI Reagent (1 mL/50–100 mg tissue), and then stored for 5 min at room temperature. Next, 0.2 mL of chloroform was added, and mixed vigorously for 15 s. The resulting mixture was stored at room temperature for 15 min and centrifuged at 12,000 g for 15 min at 4 °C. The aqueous phase was transferred to a fresh tube and the RNAs were precipitated by mixing with 0.5 mL of isopropanol. The samples were then incubated at room temperature for 10 min, followed by centrifugation at 12,000 g for 8 min at 4 °C. The RNA pellet was washed with 1 mL 75% ethanol and was centrifugated at 7500 g for 5 min at 4 °C and were dissolved in ultra-pure molecular-grade water. The quantitative PCR (qPCR) assay was carried out using the Rotor-Gene Q instrument (QIAGEN, Germantown, MD, USA) and QuantiNova® Pathogen +IC Kit (QIAGEN, Germantown, MD, USA). The final master mix (15 μL) comprised of 5 μL of 4× Reaction Mix, 6.3 μL of PCR-grade water, 1.6 μL of each primer (at 0.8 μM final concentration), and 0.5 μL of probe (at 0.25 μM final concentration) (Table 1). Then, 5 μL of the template RNA (500 ng) was added to the master mix. The cycling conditions that were used were 50 °C for 10 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s.



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