Pcr sybr premix ex taq 2

Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesized from 2 μg of total RNA with the PrimeScript RT Master Mix (Perfect Real Time; Takara). RT-PCR was performed with KOD Plus (TOYOBO). cDNA was amplified by initial denaturation at 95 °C for 3 min; 25 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; and a final elongation step at 72 °C for 5 min. PCR products were separated on 2% agarose gels. Quantitative PCR was carried out using PCR SYBR Premix Ex Taq II (Takara) and a Thermal Cycler Dice real-time system (Takara). The conditions of amplification were as follows: 10 s at 95 °C, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s, with a final 5 s at 95 °C and 30 s at 60 °C. Gene expression was normalized to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reactions were run in triplicate and repeated three times, and the results were combined to generate the graphs. We used the primers listed in Supplementary Table S1.

Gene expression was analyzed by real-time RT-PCR. Total RNA was extracted from cultured cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Two micrograms of total RNA were used to generate the first-strand cDNA with the PrimeScript RT Master Mix (Perfect Real Time; Takara, Japan). Real-time PCR was carried out using PCR SYBR Premix Ex Taq II (Takara, Japan) and a Thermal Cycler Dice real-time system (Takara, Japan) with the following conditions: 10 s at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, with a final 5 s at 95°C and 30 s at 60°C. For the DSPP gene expression, PCR condition was at 10 s at 95°C, followed by 45 cycles of 95°C for 5 s and 63°C for 30 s, with a final 5 s at 95°C and 30 s at 60°C. The reactions were run in triplicate and repeated at least three times. The primers sequence are shown in Supplementary Table S1.

Total RNA of RA and OA synovial tissues was extracted using a TRIzol™ kit (ThermoFisher Scientific, MA, USA) and reverse transcribed into cDNA according to the instructions of the first-strand cDNA synthesis kit (TaKaRa, Kyoto, Japan). Real-time polymerase chain reaction (real-time PCR) was performed using SYBR Premix Ex Taq II PCR (TaKaRa, Kyoto, Japan) on ABI Q6. The reaction conditions were as follows: predenaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s, for a total of 40 cycles. Dissolution curve analysis was performed, and each sample was run in triplicate. The reaction volume was 20 µL, including 2 µL cDNA, 0.8 µL primer, 10 µL SYBR Premix and 0.4 µL ROX, and ultrapure water was added to 20 µL. The 2^−ΔΔCt method was used for data analysis. The sequences of the primers are shown in Supplementary Table 1.