Trizol kit

The quantitative real-time PCR was carried out using TaqMan one-step PCR Master Mix (Applied Biosystems, CA, USA). Total RNA was extracted using a TRIzol kit (MDBio Inc., Taipei, Taiwan). The cDNA was reverse transcribed using 2 μg of total RNA with an oligo(dT) primer. Each cDNA (100 ng/25μl reaction) sample was mixed with sequence-specific primers and probes according to the manufacturer's instructions. Sequences for all target gene primers and probes were purchased commercially (Applied Biosystems). β-actin was used as the internal control. The cycling conditions consisted of 10 min of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s.
For miRNA detection, total RNA was extracted using a TRIzol kit (MDBio Inc., Taipei, Taiwan) and expression of miRNA was analyzed using Mir-X miRNA First-Strand Synthesis and Mir-X miRNA qRT-PCR SYBR kits (Clontech Laboratories, Inc., CA, USA). U6 snRNA was used as the internal control. The specific forward primer of miR-381 was 5′-TATACAAGGGCAAGCTCTCTGT-3′. The U6 forward and reverse primers were 5′-CTCGCTTCGGCAGCACATATACTA-3′ and 5′-ACGAATTTGCGTGTCATCCTTGCG-3′. Results were expressed as Ct values and normalized to calculate the average Ct of each sample (ΔCt), and the relative expression of miR-381 was calculated using the comparative Ct method.

Total RNA was extracted from chondrosarcoma cells using a TRIzol kit (MD Bio Inc., Taipei, Taiwan). The reverse transcription reaction was performed using 2 μg of total RNA that was reverse transcribed into cDNA using an oligo(dT) primer. RT-qPCR analysis was carried out using TaqMan^® one-step PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Total complementary DNA (100 ng/25 μL reaction) was mixed with sequence-specific primers and TaqMan^® probes according to the manufacturer's instructions. Sequences for all target gene primers and probes were purchased commercially (β-actin was used as the internal control) (Applied Biosystems). qPCR assays were carried out in triplicate using a StepOnePlus sequence detection system. The cycling conditions were 10 min of polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s.
For miRNA detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR^® RT-qPCR. U6 snRNA was used for normalization. The specific forward primer of miR-519d was as follows: 5′-CAAAGTGCCTCCCTTTAGAGTG-3′. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted as CT).

The study protocol was approved by China Medical University Hospital’s Institutional Review Board (CMUH 104-REC2-055, CMUH103-REC2-023). Written informed consent was obtained from all study participants prior to study commencement. Tumor tissue specimens were collected from patients with chondrosarcoma undergoing surgical resection procedures and human cartilage tissues were obtained from otherwise healthy OA patients undergoing knee replacement surgery; all procedures were performed in China Medical University Hospital. Total RNA was extracted from tissue using a TRIzol kit (MDBio, Taipei, Taiwan), and all unused human tissue was stored at –80 °C.