Nucelospin rna kit

Ba/F3-gp130 cells were washed 3 times with PBS and serum starved for 2.5 h. 5 nM Hyper-IL-6 or 5nM sIL-6R/sIL-11R plus 5 nM IL-6/IL-11 were incubated with 1 μg/mL sgp130Fc variant for 20 min. Then, the cells were stimulated with the mixture for 1 h. Cells were harvested and the RNA was isolated using the NuceloSpin RNA Kit (Macherey-Nagel, Düren, Germany) according to manufacturers' instructions. The RNA concentration was measured on a ScanDrop2 (Analytik Jena, Jena, Germany) and 2 μg RNA were reversely transcribed into cDNA to a final volume of 20 μL using 5 μM Oligo-dT primer and the RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA) according to manufacturers' instructions. For quantitative PCR, cDNA transcribed from 50ng RNA was analyzed using the SYBR Green Mastermix (Thermo Fisher Scientific, Waltham, USA) in a total volume of 10μL according to manufacturers' instructions. The PCR was performed on a QuantStudio 6 (Thermo Fisher Scientific, Waltham, USA). The samples were initially incubated at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The measurements were performed in triplicates. SOCS3 expression was normalized to GAPDH and the relative gene expression levels were calculated using the 2^−ΔΔCt method.