Sybr green real time pcr master mix kit

Total RNA was isolated by using RNAiso Plus (TaKaRa) and then synthesized into cDNA by using the cDNA Synthesis Kit (Beyotime, China). Real-time PCR was performed with a SYBR Green Real-time PCR Master Mix kit (Beyotime, China). Real-time PCR was performed on an Applied Biosystems, StepOnePlus Instrument (USA) with the following cycling program: 95°C for 5 min pre-degeneration, followed by 40 cycles of amplification at 95°C for 15 min and 60°C for 60 s, then 95°C for 15 s. The gene expression level was normalized by GADPH, and ΔΔCT was calculated by referring to the control group. The primers used are listed in Table 1.

After 3 days of incubation with culture media containing different [Ca²⁺]_, chondrocytes mRNA was collected and purified using RNAiso Plus (TaKaRa, Dalian, China). The mRNA was dissolved in DEPC-treated water and quantified using Nanodrop (ThermoFisher, Carlsbad, CA, USA). Then, mRNA was transcribed to cDNA using the cDNA Synthesis Kit (Beyotime, Shanghai, China). The gene expression levels were detected with an SYBR Green real-time PCR Master Mix kit (Beyotime, Shanghai, China) and performed on an Applied Biosystems, StepOnePlus Instrument (Thermofisher, Carlsbad, CA, USA). The gene expression levels were normalized by β-actin; 2^−∆∆ct was calculated by referring to the 1.25 mM calcium ion group. All the primers were shown in Table S2.