Rapid site directed mutagenesis kit

The pmirGLO Dual-Luciferase miRNA Target Expression Vector and Dual-Luciferase® Reporter Assay were purchased from Promega. Using bioinformatics to analyze and predict the binding site of miRNA-188-3p to APOC1P1-3, we designed primers and cloned the target fragment. The fragment was inserted into the pmirGLO Dual-Luciferase miRNA Target Expression Vector by molecular cloning, and the recombinant reporter plasmid and the target microRNA mimic were co-transfected into the target cells. The luciferase was extracted and its activity was tested. The mutated fragment was ligated into the dual luciferase reporter gene vector, and the target microRNA mimic was co-transfected with the target cell for luminescence detection. The DNA purification kit、Glue recovery kit and Rapid site-directed mutagenesis kit were purchased from TIANGEN. Endonuclease XhoI、SalI and T4 ligase were from NBE.

The FUNDC1 3′-UTR or Lnc049808 sequence (including the miR-101 binding site) was inserted into the pGL3 luciferase vector (Promega, USA) to construct the luciferase reporter vector. A rapid site-directed mutagenesis kit (TIANGEN, China) was used to generate mutations in the seed region of miR-101 as a mutation control. A dual-luciferase reporter gene assay system (Promega) was used to measure luciferase activity.