Real time PCR (qPCR) assay

The primary chondrocytes were divided into three groups as follows: NC group was the normal control cultured for 30 h with no treatment; TNF-α group was the model group pre-treated with TNF-α (10 ng/ml) for 6 h and then cultured for 24 h with no further treatment; and TNF-α+PL group was the treatment group pre-treated with TNF-α (10 ng/ml) for 6 h and then treated with PL (derived from 10⁷ platelets) for 24 h. Total RNA was extracted with TRIzol reagent and quality controlled by NanoDrop2000 spectrophotometer (Thermo Scientific, USA) and agarose gel electrophoresis. The quality of all RNA samples was verified with good purity and integrity before use. Then, the reverse transcription was conducted to produce cDNA. As previously applied, the final qPCR reaction system was 20 μl, comprising 10 μl SYBR^® Premix Ex Taq II (Tli RnaseH Plus), 0.4 μl PCR Forward Primer, 0.4 μl PCR Reverse Primer, 1 μl template cDNA and 8.2 μl ddH₂O, and the qPCR reaction conditions were as follows: 95°C for 5 min for initial denaturation, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing and extension at 60°C for 30 sec [60]. The qPCR assay was performed on an ABI QuantStudio^TM 7 Flex Real-Time PCR System (Applied Biosystems; Thermo Scientific, USA). β-Actin was used as the reference gene and 2^{-ΔΔ CT} method was applied to measure the relative mRNA expression (Table 1).