Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA was extracted from human synovial fibroblasts (1×10⁶) that had previously undergone different treatments, or osteochondral samples from control and OA rabbits, using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). One Step SYBR PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) and an iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) were used for PCR. The temperature protocol used for RT was 42°C for 5 min followed by 95°C for 10 sec. The following thermocyling conditions were used for qPCR: 40 cycles at 95°C for 5 sec followed by 60°C for 30 sec; followed by a final termination step at 4°C for 10 min. Relative gene expression was determined by the 2^−ΔΔCq method (19) and expression level of the GAPDH gene from the same samples was used as an internal control. Specific oligonucleotide primers for S100B, TNF-α, IL-1β, FGFR1 and GAPDH are listed in Table II.