Rnase free dnase set 50

Total RNA was isolated from PBMCs using the RNeasy plus mini kit from Qiagen (Catalog No. 74134) as per manufacturer’s protocol. Quantification of IL-32 isoforms (α, β, γ, D, ε and θ) was carried out using one-step SYBR Green quantitative real-time PCR, which was performed on the LightCycler 480 II (Roche) apparatus using QIAGEN reagents (RNase-Free DNase Set (50); Cat. No. 79254). Real-time qRT-PCR was performed in duplicates using 25ng RNA per reaction. No-template controls were included in parallel for each gene master mix. Gene expression for each individual isoform was normalized to the housekeeping gene β-glucuronidase. Primer sets for IL-32α, β, ε (C) and β-glucuronidase have been described earlier ²⁷ , while primer sets for IL-32 γ and θ were designed based on γ and θ transcript reference sequences as summarized in Supplemental Table 2.

Kidneys from 5 mice at each point were thawed in Trizol (Invitrogen, Carlsbad, CA, USA) and broken with 0.5 mm zirconia/silica beads (Biospec) using a Bead Beater apparatus (Biospec Products, Inc., Bartlesville, OK, USA) at high-speed setting (3 pulses of 12–15 sec). Total RNA was extracted using Trizol reagent per the manufacturer’s instructions. Contaminating gDNA was removed using RNeasy mini spin column (RNeasy mini kit, Qiagen Science, MD, USA) and RNase-free DNase set (50) (Qiagen GmbH, Germany), according to the manufacturer’s instructions. The quantity and quality of the extracted RNA were assessed by A260 nm/A280 nm ratio (NanoDrop 1000 spectrophotometer, Thermo Scientific), as well as by electrophoresis through a 1.5% agarose gel stained with ethidium bromide for the presence of intact 18S and 28S ribosomal RNA bands, visualized by UV transillumination at 302 nm. RNA purity was determined using A260:A280 ratios and the extinction coefficient for RNA to quantify the amount of RNA present [24 (link)].