Taqman gene expression assays and reagents

Total RNA was extracted from homogenized kidney tissue or purified cells using TRIzol reagent (Invitrogen) or the RNeasy^® Micro Kit (Qiagen), respectively. The QuantiTect Reverse Transcription Kit (Qiagen) was used for cDNA synthesis according to the manufacturer’s protocol. Transcripts were quantified by quantitative reverse transcription PCR (RT-qPCR) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems) with predesigned TaqMan Gene Expression Assays and reagents according to the manufacturer’s instructions (Applied Biosystems), alternatively with predesigned SYBR Green master mixes (Thermo Fisher Scientific) and custom-designed primers (Supplementary Table 1). Probes with the following Applied Biosystems assay identification numbers were used: Mm99999915_g1 (GAPDH), Mm01288386_m1 (IL1B), Mm00446190_m1 (IL6), and Mm00443258_m1 (TNF). For each sample, mRNA abundance was normalized to the amount of GAPDH and is presented as fold gene expression (2^(ΔΔCt)).

RNA was extracted with the RNeasy mini kit (Qiagen, Milan, Italy), and cDNA was transcribed using a reverse transcription kit (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions. The real-time PCR reactions were performed in multiple replicates and run on an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Warrington, UK) with predesigned TaqMan Gene Expression Assays and reagents (Applied Biosystems, Warrington, UK), according to the manufacturer's instructions.
Regarding the longitudinal study, the procedures of RNA extraction and reverse transcription were performed immediately after the sampling, while the cDNA amplification of all samples were performed simultaneously. mRNA onset (ratio between the value of cytokine gene expression produced in response to the IFX and the value of cytokine gene expression produced in response to medium alone) ≥2 was considered positive.

For evaluation of mRNA expression (IFN-γ, IL-13, IL-17, IL-10, IL-12A, EBI-3 and Foxp3), 1 × 10⁶ cells were cultured for 12 h in complete medium and 5% heat-inactivated human serum AB in 48-well round-bottomed microwell plates with or without RTX or TCZ (50 μg/ml).
RNA was extracted with the RNeasy mini kit (Qiagen, Milan, Italy), and cDNA was transcribed using a reverse transcription kit (Applied Biosystems, Warrington, UK) according to the manufacturer’s instructions. The real-time PCR reactions were performed in multiple replicates and run on an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Warrington, UK) with predesigned TaqMan Gene Expression Assays and reagents (Applied Biosystems, Warrington, UK), according to the manufacturer’s instructions as described^{16 (link),17 (link)}.
mRNA ratios (between the value of cytokine gene expression produced in response to the drugs and the value of cytokine gene expression produced in response to medium alone) ≥ 2 were considered as positive.