Sybr premix ex taq 2

The total RNAs were extracted from cultured cells using TRIzol reagent (Takara Bio Inc., Dalian, China) according to a previously described method [27 (link)]. Six hundred ng of total RNA was reverse-transcribed into first-strand cDNA using the Prime RT Master Mix kit (Takara Bio Inc., Dalian, China). cDNA samples containing an equivalent amount of total RNA were quantified using SYBR Premix EX Taq II (Yeasen Biotech Co., Shanghai, China) with specific primer pairs and subjected to the CFX Connect™ Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). The specific primer pairs used are listed in Table S3.

5 × 10⁵ CD226⁺Lin⁻CD117⁻Sca‐1⁺ MSCs and CD226⁻Lin⁻CD117⁻Sca‐1⁺ MSCs were sorted, and total RNA was harvested using Trizol reagent (Sigma, T9424). RNA (1 μg) was reverse transcribed using PrimeScript RT Master Mix (Yeasen, 11141ES). Then RT‐qPCR was performed using SYBR Premix Ex Taq II (Yeasen, 11184ES) by Bio‐Rad CFX96 Touch™ real‐time PCR detection system with specific primers. The primer sequences are listed in Table S2. The relative amount of IL‐6, RANKL, M‐CSF and CD200 mRNA was determined using 2^−ΔΔCt method and normalized to the 18s housekeeping gene. The experiment was repeated three times.

In this study, we use TRIzol reagent (Invitrogen, Carlsbad, CA, United States) to extract total RNA from human nucleus pulposus cells and rat nucleus pulposus tissues.
Total RNA was extracted from human nucleus pulposus cells or rat nucleus pulposus tissues by using the TRIzol RNA isolation protocol (Invitrogen, Inc., Carlsbad, CA, United States). AG Reverse Transcription Kit (Accurate biotechnology, Changsha, Hunan, China) was used to reverse transcribe total RNA into cDNA and SYBR Premix ExTaq II (Yeasen Biotechnology, Shanghai, China) SYBR was used for the reactions of qPCR. Then we used a ABI 7500 sequencing detection system (Applied Biosystems, Foster City, CA, United States) to measure the reactions and applied the β-actin as a control RNA expression level. Data were analyzed by GraphPad Prism (GraphPad Prism Software 6.0, United States). The primers we used in the experiments: β-actin, AGA​GCT​ACG​AGC​TGC​CTG​AC, PLK1, CAC​CAG​CAC​GTC​GTA​GGA​TT, MMP3, CCT​ACA​AGG​AGG​CAG​GCA​AG, MMP13, TCG​GCC​ACT​CCT​TAG​GTC​TT, COL2A1, CCA​GAT​GAC​CTT​CCT​ACG​CC, ADAMTS4, AAC​GTC​AAG​GCT​CCT​CTT​GG, ADAMTS5, CCG​GAG​CCA​CTG​CTT​CTA​TC, aggrecan, GGG​ACC​TGC​AAG​GAG​ACA​GAG, SOX9, GCT​CTG​GAG​ACT​TCT​GAA​CGA, p53, CCA​GGA​TGT​TGC​AGA​GTT​GTT​A, p21, TAT​TTT​GTC​CTT​GGG​CTG​CCT, p16, CTT​CGG​CTG​ACT​GGC​TGG.