RNA extraction and gene expression of chondrocytes

The chondrocytes were collected and total RNA was extracted using RNeasy Protect Mini kit (74104, QIAGEN, Germany). Total RNA yield and RNA quality were detected by spectrophotometer (NanoDrop™ 2000, Thermo Fisher Scientific, USA). RNA samples showed an A260/280 ratio between 1.8~2.0 and an A260/230 ratio between 2.0~2.2 were used for reverse transcription quantitative polymerase chain reaction (RT-qPCR). The first strand complementary DNA (cDNA) was synthesized from RNA and SuperScript™ III First-Strand Synthesis System (18080-051 Invitrogen, USA) according to the instructions provided by the manufacturer. The volume of the PCR Mix of single reaction was 20 μl containing 1 μl of primer solution, 9 μl of cDNA and 10 μl of 2× TaqMan Universal PCR Master Mix (4304437, ABI, USA). TaqMan Gene Expression Assays (Life Technology, USA) were used for gene expression analysis. The target genes of RT-qPCR are summarized in Table 1. RT-qPCR was performed using an ABI PRISM 7900HT Sequence Detection System and Sequence Detection Software 2.2.2. The target genes were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative mRNA expression of each target gene was determined using the ^∆∆Ct method.

Primers used in this study