Chromo4 four color real time pcr detection system

Real-time quantitative PCR (qPCR) was performed on a Chromo4 four-color real-time PCR detection system (Bio-Rad). qPCR for each sample was performed twice in triplicates using a 20-μl reaction system containing 50 ng initial total RNA. Both annealing and extending temperature were set to 60°C. Forty PCR cycles were run and the melting curves were recorded. The primers used are as follows: 5′-GAAGAGAGGGAACCACAGCA-3′ (sense) and, 5′-TTGCCTGTTAAATGGGCCAC-3′ (anti-sense) for FasL; 5′-TCATCGCGGTATTCGGTTCG-3′ (sense) and 5′-CTTCACCTCCGTGATTGCCT-3′ (anti-sense) for Bim; 5′-ACGGGGTTAGCGGAGCAA-3′ (sense) and 5′-ATGTCCATTCCATGAAGTCAGC-3′ (anti-sense) for p27^Kip1; 5′-CCTCCTTGGAAAGCAAA CAGTAAA-3′ (sense) and 5′-ACACTCACTGGCT TTTCATCTTC-3′ (anti-sense) for cyclin A2; 5′-AGAT CTGGCACCACACCTTCT-3′ (sense) and 5′-CTTTG ATGTCACGCACGATTT-3′ (anti-sense) for β-actin. All other parameters for the reaction system and the PCR program were set according to the manufacturer's protocol for the SYBR PrimeScript RT-PCR kit (Takara, Dalian, China). The specificity of PCR was determined by a following melting curve analysis. The relative quantification of gene expression was analyzed by the 2^−ΔΔCt method.

Total RNA was isolated from chondrocytes cultures using the TRIzol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocol. Then, the total RNA products were immediately transcribed by reverse transcription (RT) into cDNA by using a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Polymerase chainreaction (PCR) amplification was performed in a Chromo4 Four-Color Real-Time PCR Detection System (Bio-Rad) by using the SYBRR Premix Ex Taq II kit (TaKaRa). Primer sequences (Life Technologies) for each gene used in this study are shown in Table 1.