Fluorescence rt qpcr kit

RIPA lysis buffer, BCA Protein Assay kit and 5X loading buffer were purchased from Beyotime Institute of Biotechnology. Rabbit polyclonal anti-FXR (cat. no. ab28676) and anti-TGR-5 (cat. no. ab72608) were purchased from Abcam. The mouse monoclonal antibody to β-actin (cat. no. sc517582) was purchased from Santa Cruz Biotechnology, Inc. The goat anti-Rabbit IgG (cat. no. BS13278) and goat anti-Mouse IgG (cat. no. BS12471) were purchased from Bioworld Technology, Inc. eECL Western Blot kit (cat. no. CW0049A) was purchased from CoWin Biosciences. RevertAidFirst Strand cDNA Synthesis kit was purchased from Thermo Fisher Scientific, Inc. SYBR Premix Ex Taq was purchased from Thermo Fisher Scientific, Inc. The primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The fluorescence RT-qPCR kit was purchased from Takara Biotechnology, Co., Ltd. TRIzol^® and the immunohistochemistry kit were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The 5% bovine serum was purchased from Wuhan Boster Biological Technology. Ltd.

Total RNA was extracted according to the Trizol instructions, and the RNA was converted into cDNA using the PrimeScript RT kit (Takara, Japan). The PCR reaction system was prepared according to the instructions of the fluorescence RT-qPCR kit (Takara, Dalian, China). Samples were run on a real-time fluorescence RT-qPCR instrument system (ABI 7500; ABI, Foster City, CA, USA) and subjected to fluorescence RT-qPCR. Using GAPDH and U6 as internal references, the relative expression of each target gene was calculated by the 2^−ΔΔCt method, and three replicate wells were set for each sample. ΔΔCt = ΔCt experimental group − ΔCt control group, ΔCt = Ct target gene − Ct internal reference. The primer sequences are shown in Table 1.