Nucleospin rna protein mini kit

RNA from cells after 4 d of drug treatment was isolated using the NucleoSpin RNA/protein mini kit (Macherey-Nagel, Allentown, PA, USA, 740933.50) according to the manufacturer’s instructions. The concentration of isolated RNA was measured using a Nanodrop 2000c Spectrophotometer (Thermo Scientific). Complementary DNA (cDNA) was synthesized from RNA using a QuantiTect reverse transcription kit (Qiagen, Germantown, MD, USA, 205313) following the manufacturer’s instructions. A SYBR-based PowerTrack master mix (Thermo Fisher Scientific A46012) was used to perform RT-qPCR on a QuantStudio 6 Pro Real-Time PCR system (Thermo Fisher Scientific) in 10 μL reactions. The thermal protocol was as follows: 95 °C for 2 min, then 40 cycles of 95 °C for 15 s and 60 °C for 60 s. PCR primer sequences are listed in Table 2 and were the same as used in Larson et al., 2019 [17 (link)]. Fold change in gene expression was determined using the ΔΔCT method [49 (link)] with GAPDH as the housekeeping gene.

RNA from monocytes, macrophages and HUVEC was isolated using the Nucleospin RNA/Protein mini kit (Macherey-Nagel). Reverse transcription of total RNA was performed using iScript (Bio-Rad) and PCR assays were performed in duplicates using SYBR green (Bio-Rad) and specific primers (Integrated DNA Technologies; Supplementary Table S2) with a CFX96 Touch-Real-Time PCR system (Bio-Rad). Relative gene expression was normalized to B2M and GAPDH housekeeping genes expression. The relative expression and quantity (RQ) of mRNA were calculated using the formulas 2^{–ΔCtx 1000} or 2^–ΔΔCt, respectively. IFN signature was evaluated in PBMCs from HC and patients with SLE using expression data of IFI44L, ISG15, IFIT2, IFIT3 and MX1 and obtaining a Z score to classify the patients (25 (link)).