siRNA Transfection Technique

RA FLS were cultivated in classic medium antibiotic free for 24 hours and transfected at 80% confluency, before transfection FLS were rinsed using siRNA transfection medium (Santa Cruz Biotechnology, Dallas, TX, USA). RA FLS were transfected by adding YAP siRNA or control siRNA (Santa Cruz Biotechnology) at 7.5 µg/ml in siRNA transfection reagent (Santa Cruz Biotechnology; diluted 6/100 in transfection medium) for 6 hours at 37°C in a CO₂ incubator. DMEM containing 20% FBS and 2% PS was added to the wells overnight (achieving a ½ dilution of the added medium). Medium was then replaced with or without TNF and IL-17 for 48 hours before extraction. On soft substrate, RA FLS siRNA transfection was noneffective due to bovine fetal serum retention in the gel.