Real time pcr kit

Total RNA from tissues or cells was extracted with TRIzol Reagent. The concentration of total RNA was determined by the spectrophotometer Q5000 (Quawell Technology, USA) and then reverse-transcribed into cDNA by using a reverse transcription kit (Vazyme Biotech, China). qPCR was carried out using a real-time PCR kit (GenePharma, China) in accordance with the manufacturer's instructions. The 2^–ΔΔCt method was utilized to calculate the relative expression of genes. MiR-30a was normalized to U6 snRNA, and the IL-1β and IL-6 expression was normalized to GAPDH. The primers are listed in Supplementary Table 1.

Baicalin was obtained from Nanjing Zelang Medical Technology. LPS, dimethyl sulfoxide (DMSO), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), indomethacin (INDO), dexamethasone (DEX), aminoguanidine (AG, iNOS inhibitor), NS-398 (COX-2 inhibitor), and pyrrolidine dithiocarbamate (PDTC, IκBα, and NF-κB P65 inhibitor) were purchased from Sigma-Aldrich. NO and iNOS assay kits were purchased from Nanjing Jiancheng Bioengineering Institute. Real-time PCR kit was purchased from Shanghai Gene Pharma. PGE2 and IL-1β ELISA kits were purchased from R&D Systems. MAPK family antibody sampler kit, phosphorylated MAPK family antibody sampler kit, and primary antibodies against β-actin, COX-2, IRAK1, IRAK4, MyD88, and NF-κB P65 were purchased from Cell Signaling Technology. Interleukin-1 receptor-associated kinase 1/4 inhibitor (IRAK1/4 inhibitor), TLR4, P-IκBα, and IκBα antibodies were purchased from Santa Cruz Biotechnology. MTS510 (TLR4 inhibitor) was purchased from eBioscience. SB203580 (P38 MAPK inhibitor), SP600125 (JNK inhibitor), ST2825 (MyD88 inhibitor), and U0126 (ERK inhibitor) were purchased from the Beyotime Institute of Biotechnology. RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco.