Sybr green pcr matrix mix

After treated with TNF-α and Atsttrin, the human NP cells were disintegrated with TRIzol reagent (Takara Bio, Japan). After total RNA was extracted then reverse transcribed to cDNA according to the manufacturer’s instructions of cDNA Synthesis Kit (GeneCopoeia, Inc. USA). Real-time PCR reactions were carried out on Roche LightCycler (Roche, USA) utilizing a SYBR Green PCR Matrix Mix (Toyobo, Japan). The nucleotide sequences of MMP-13, COX-2, iNOS and GAPDH primers are listed in Table 1.

Mouse nucleus pulposus tissues (2-3 mg) and nucleus pulposus cells were treated with TNF-α and lysed with TRIzol reagent (Takara Bio, Japan). A total of 1 μg of RNA was used for reverse transcription by a cDNA synthesis kit (GeneCopoeia, Inc., USA). RT-PCR was performed in 10 μl of SYBR Green PCR matrix mix (Toyobo, Japan) on a thermal cycler (Bio-Rad, Hercules, USA). Cycle parameters were as follows: 95°C for 1 min, 40 cycles (95°C for 15 s, 60°C for 15 s, and 72°C for 45 s), and 72°C for 5 min. The RT-PCR results were calculated using the 2^-ΔΔCt method. The primers (FSTL-1, COX-2, MMP-13, ADAMTS-5, β-actin, and iNOS) were designed based on published sequences of these genes and listed in Table 1 [29 , 30 (link)].