Gene Expression Analysis of Chondrocytes

RNA isolation from monolayer chondrocyte pellets was performed using NucleoSpin RNA/Protein kit (Machrey Nagel, Düren, Germany) according to the manufacturer’s instructions. Synthesis of cDNA was performed using qScript cDNA Supermix (Quanta Biosciences, VWR, Darmstadt, Germany). For gene expression analysis of different AR subtypes and the IL-1β receptor (IL-1βR) on chondrocytes at day 0 and day 7, reverse transcription PCR was used (Taq PCR Master Mix kit, Qiagen, Hilden, Germany). PCR products were run on a 1.8% (wt/vol) agarose gel, stained with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA). In addition, TH gene expression was quantified in order to consider possible autocrine effects. GAPDH served as housekeeping gene. In addition, gene expression changes of ARs and ECM-related genes (SOX9; COL1A1; COL2A1; COL10A1; COMP, ACAN; MMP13; ADAMTS-4; ADAMTS-5) after seven days of monolayer culture were analyzed using Quanta PerfeCta SYBR Green FastMix (Quanta Biosciences, VWR, Darmstadt, Germany) in qTOWER3 Thermocycler (Analytik Jena, Jena, Germany). Relative gene expression was determined by the ∆∆Ct method using qPCR3.2 software (Analytik Jena) [52 ]. Human RPII served as housekeeping gene [53 (link),54 (link)]. All primers were synthesized by Thermo Fisher Scientific (Table 2).