*4.4. Gene Expression Analysis*

Total RNA was extracted from IFP away from the synovial membrane using QIAMP mini kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. First-strand cDNAs were synthesized from equal amounts of total RNA using random primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative real-time PCR (qRT-PCR) for adipokines (leptin, adiponectin, peroxisome proliferative activated receptor gamma2 [PPARγ], fatty acid binding protein 4 [FABP4]), cytokines (IL-6, TNF-α, monocyte chemotactic protein 1 [MCP-1]), vascular endothelial growth factor (VEGF), chemokines (C-X-C motif chemokine ligand 8 [CXCL8]), genes involved in extracellular matrix remodeling (collagen type I [COLI], collagen type III [COLIII], and collagen type VI [COLVI], transmembrane glycoprotein NMB [GPNMB], inter-α-trypsin inhibitor heavy chain 5 [ITH5], serine proteinase inhibitor 2 [SERPIN2]), and transforming growth factor (TGF-β) was performed using Sybr-Green fluorophore and specific primers (Table S1, Supplementary Materials). Reaction efficiency was established for each set of primers and for an endogenous unregulated reference gene (18 s), after quantification of six different dilutions of the cDNA pool and calculated from the slope according to the equation E = 10−1/slope [42]. qRT-PCR was performed in triplicate for each gene and carried out in duplicate for each sample by DNA Engine Opticon 2 (MJ Research, Waltham, MA, USA). Melting curves confirmed the specificity of the amplification signal target of our gene. Data were calculated using the comparative (ΔΔCt) method as the ratio of each gene to the expression of the housekeeping gene and are represented as fold-change versus controls.
