Cfx384 real time thermal cycler

Total RNA extraction was performed by Trizol (Life Technologies). Reverse transcription was performed using random priming and Maxima First Strand cDNA Synthesis Kit (ThermoFisher), according to manufacturer’s guidelines. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed by Absolute SYBR Green mix (Thermo Scientific) in a CFX384 real-time thermal cycler (BioRad). Variations in input RNA were corrected by substracting PCR threshold cycle values obtained for GAPDH.

Quantification of microRNA expression levels were performed by RT-qPCR using the miRCURY LNA™ Universal RT microRNA PCR system and specific microRNA LNA™ PCR primer sets (Exiqon, Vedbaek, Denmark). Specific microRNA target sequences are shown in Table 1. cDNA was diluted 80× and run in accordance with Exiqon’s instructions manual on a CFX384 Real-Time thermal cycler (Biorad, Hercules, California, USA). Each sample was run in duplicate, and negative controls with no-template and blanks as well as a spike-in, UniSp6, were assayed in each run.

Total RNA was extracted with Trizol (Life Technologies). Reverse transcription was performed using random priming and Maxima First Strand cDNA Synthesis Kit (ThermoFisher), according to the manufacturer’s guidelines. Quantitative real-time polymerase chain reaction (PCR) was performed using Absolute SYBR Green mix (Thermo Scientific) in a CFX384 Real-time thermal cycler (BioRad). Variations in input RNA were compensated for by subtracting the PCR threshold cycle values obtained for GAPDH.

Total RNA was extracted from kidney samples and cells using TRI reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using an M-MLV reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA) and quantitative PCR carried out using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on a CFX384 Real Time thermal cycler (Bio-Rad). The target genes were as follows: CCL2 (C-C motif chemokine ligand 2, also known as monocyte chemoattractant protein 1: MCP-1), CCL3 (C-C motif chemokine ligand 3, also known as macrophage inflammatory protein-1α: MIP-1α), CCL5 (C-C motif chemokine ligand 5, also known as regulated upon activation, normally T-expressed, and presumably secreted: RANTES), CXCL2 (C-X-C motif chemokine ligand 2, also known as macrophage inflammatory protein 2α: MIP-2α), CXCL13 (C-X-C motif chemokine ligand 13), TNF-α (tumor necrosis factor-α), iNOS (inducible nitric oxide synthase), Nox2 (NADPH oxidase 2), collagen types 1a1 and 3a1, TGF-β1 (transforming growth factor β1), CD68 (cluster of differentiation 68), CD86, and CD206. Target gene expression was normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA expression. Primer sequences are shown in Table S1.