Ag rnaex pro rna kit

Total RNA was extracted from the cell lines using AG RNAex Pro RNA Kit (AG21101, Accurate Biotechnology, Changsha, Hunan, China). Subsequently, cDNA was synthesized and reverse transcribed using the Evo M-MLV RT Kit with gDNA Clean for RT-qPCR (AG11705, Accurate Biotechnology, Changsha, Hunan, China). Real-time polymerase chain reaction (RT-PCR) was performed with the SYBR Green Pro Taq HS premixed qPCR kit (AG11701, Accurate Biotechnology, Changsha, Hunan, China), and expression levels were calculated using the 2^{^(−ΔΔCt)} method. mRNA expression was normalized to the expression level of β-actin mRNA. All primers were provided by Accurate Biotechnology Co., Ltd. (Changsha, Hunan, China), and the detailed primer sequences are shown in Supplementary Table S1. All data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Quantitative real‐time polymerase chain reaction (qRT‐PCR) reagents and supplies are AG RNAex Pro RNA kit, SYBR Green Pro Taq HS kit, Evo M‐MLV reverse‐transcription kit (removal gDNA reagent) and Rox and were purchased from Accurate Biotechnology (Hunan) Co. Ltd. People's Republic of China. Western‐blotting reagents and supplies are Rabbit Anti‐Ngb, Polyclonal Antibody (bs‐1859R), Rabbit Anti‐HIF‐1, Alpha Polyclonal Antibody (bs‐0737R), Rabbit Anti‐beta‐Actin (Loading Control), Polyclonal antibody (bs‐0737R) and goat anti‐rabbit IgG/HRP(bs‐0295G‐HRP) and were purchased from Bioss Co. Ltd. People's Republic of China. RIPA tissue or cell rapid lysate was purchased from Bio topped, and 0.22μm polyvinylidene difluoride filter (PVDF) membranes, 4 × protein loading buffer (DTT), Rainbow 245 broad‐spectrum protein marker (11‐245KD) and ECL hypersensitivity luminescent solution were purchased from Solarbio Co. Ltd. People's Republic of China. Immunohistochemical reagents and supplies are immunohistochemical staining kit and HRP‐DAB kit and were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. People's Republic of China.