Hairpin ittm microrna qpcr quantitation kit

RNA was extracted by using TRIzol (Invitrogen). The extracted RNA was washed and resuspended in nuclease-free water before being treated with DNase I. Total RNA was quantified and its purity was evaluated using a Nanodrop 2000 (Thermo Fisher Scientific). The mRNA reverse transcription kit (Takara) and TaqMan reverse transcription kit (Life Technologies) were used for reverse-transcribing mRNA and miRNA, respectively, as per manufacturer’s instructions.
Universal SYBR Green Master mix (Applied Biosystems) was used to perform quantitative PCR (qPCR) for analyzing the mRNA expression, while Hairpin-itTM microRNA qPCR Quantitation Kit (GenePharma) was applied to detect miR-214 in RA-FLS. All qPCR experiments were completed on a 7500 real-time PCR system (Applied Biosystems); GAPDH and U6 small nucleolar RNA (snoRNA) were used as endogenous controls for normalizing the expression of the mRNA and miRNAs, respectively.

qPCR and Western blot were performed as previously reported 19 (link). qPCR was carried out using the Hairpin-it^TM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and LightCycler 96 (Roche, Germany). U6 snRNA was used as an internal control for miR-92b, and the mRNA levels of PTEN, TNF-α and IL-6 were normalized to GAPDH. The 2^-ΔΔCt method was employed to calculate relative expression of each gene. For Western blot analysis, the protein blots were incubated with following antibodies: rabbit anti-TLR4 (1:1000, 7074, Cell Signaling Technology, USA), rabbit anti-phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (1:1000, 4764, Cell Signaling Technology, USA), rabbit anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), rabbit anti-Bax (1:200, bs-0127R, Bioss, China), rabbit anti-Bcl-2 (1:200, bs-4563R, Bioss, China), rabbit anti-phospho-PI3K (1:200, bs-3332R, Bioss, China), rabbit anti-PI3K (1:1000, ab227204, Abcam, USA), rabbit anti-phospho-AKT (1:1000, ab8932, Abcam, USA), rabbit anti-AKT (1:500, GB111114, Servicebio, China), rabbit anti-β-catenin (1:5000, ab196204, Abcam, USA). The optical density (OD) value of each protein band was quantified using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics Inc., USA) and normalized to β-actin.