4.4.3. Gene Expression Analysis of IVD Cells

RT-qPCR was performed to analyze the expression of target genes (Table 6). The cells were lysed with 1 mL Trizol (Genezol, GZR200, Geneaid biotech, New Taipei City, Taiwan) and RNA was isolated according to the manufacturer's recommendations. Briefly, after adding chloroform (132950, Sigma), the samples were mixed well for 15 s, left for 5 min at RT, and centrifuged (12,000× *g* for 15 min). Supernatants were carefully transferred to new RNase free tubes, 500 μL of isopropanol (20842, VWR chemicals, Fontenay-sous-Bois, France) was added and mixed well. After 5 min, the samples were centrifuged again (12,000× *g* for 15 min). The pellets were washed with 75% ethanol (1.00983, Merck) at 7500× *g* for 5 min and then dissolved in RNase free water (10977, Invitrogen, Carlsbad, CA, USA). The purity and concentration of the resulting RNA were measured using the NanoDrop (ND-1000, Thermo Fisher Scientific). 1 μg of total RNA was reverse-transcribed to cDNA using a reverse transcription kit (4374966, Applied Biosystems, Foster City, CA, USA). qPCR of the mixture of primers/probes (Table 6) and master mix (4367846, Applied Biosystems) was performed on the CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA). The amplification program was as follows: heating at 95 ◦C for 10 min; 40 cycles of 95 ◦C for 1 s and 60 ◦C for 20 s. The relative expression level was calculated by the dd*C*<sup>t</sup> method. For normalization purposes, the samples with undetectable expression were assigned *C*<sup>t</sup> value 40. The results are shown as fold change, relative to control or relative to cytokine treatment.

**Table 6.** Target genes and assay identification (ID) numbers of corresponding TaqMan primers (TaqMan Gene Expression Assays; Thermo Fisher Scientific). TRP = transient receptor potential; MMP = matrix metalloproteinase; TIMP = tissue inhibitor of matrix metalloproteinase; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs; COX-2 = cyclooxygenase-2; NGF = nerve growth factor. IL-6 = interleukin 6; IL-8 = interleukin 8; HKG = housekeeping gene.

