Tb green premix ex taq 2 tli rnase h plus kits

Total RNA was extracted from cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. One thousand nanograms of total RNA was used for each reaction of reverse transcription by using PrimeScript RT reagent Kit (Takara) and then subjected to real-time PCR using TB Green Premix Ex Taq II (Tli RNase H Plus) kits (Takara) on an Applied Biosystems 7500 Fast Real-Time PCR System (ABI). Each sample was performed in triplicate, and all results were normalized to the expression of being normalized to β-actin (ACTIN). Data was analyzed using the 2^–ΔΔCt method against β-actin. Results were expressed as fold changes compared to the control. The sequences of PCR primers were listed in Supplementary Table 1.

Clean Sprague-Dawley (SD) rats were bought from the Animal Experimental Research Center under the Shanghai Jiao Tong University Affiliated Sixth People's Hospital (SJTU 6# Hospital). Fetal calf serum (FCS), penicillin-streptomycin (PS), Opti-MEM medium, D-MEM/F-12 medium, and FCS were all bought from Gibco Company (USA). Thermo Fisher Scientific Company sold a Lipofectamine 2000 kit and an alkaline phosphatase (ALP) activity detection kit (USA). Methyl thiazolyl tetrazolium (MTT) kit was supplied by Beijing Solarbio Science & Technology Co., Ltd. Osteoblast mineralized nodule staining reagents and Dual-Lumi™ dual-luciferase reporter (DLR) gene kit were from Beyotime Biotechnology Co., Ltd. (Shanghai). PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) kits were from Takara Biomedical Technology (Beijing) Co., Ltd. GAPDH, PTEN, PI3K, p-PI3K, AKT, p-AKT first antibodies, and horseradish peroxidase- (HRP-) labeled IgG second antibody were purchased from Abcam Company (UK).