Dual luciferase reporter gene detection kit

The wild-type and mutant 3’UTR DNA fragments of JNK1 covering the predicted binding sites of miR-33-5p were successfully replicated. The psiCHECK™-2-JNK1-3’UTR (wild type and mutant) vector was constructed by combining the luciferase vector psi-CHECK™-2 (Promega, Madison, WI, USA) with JNK1-3’UTR (wild type and mutant). Similarly, we cloned the CDS of JNK1 or Lnc90386 into the pcDNA3.1 vector. The primers are described in Table 1.
Dual-luciferase reporter assay is detailed in our previous reports (16 (link)). In a nutshell, when cells reached 80-90% confluence, using Lipofectamine 2000 (Invitrogen Life Technologies, USA) each co-transfected cells with wild-type or mutant reporter plasmid (200 ng) and 10 pmol of the indicated RNA oligonucleotides. Then, the luciferase activity in each group was detected by using an automatic microplate reader (Bio-Rad, Hercules, CA, USA) in accordance with the dual luciferase reporter gene detection kit instructions (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

The regulatory role of miR-146 on HDMCP was tested with dual luciferase report system. Firstly, the normal gene fragment containing 3’ UTR region of HDMCP wide type and the specific dual-luciferase miRNA target expression vector-pmirGLO (Promega, the USA) were both cut with SacⅠand SalⅠ. These two segments were then connected with T4 DNA ligase (Fermentas, Lithuania) at 22℃ for 2h. Secondly, the combined vector pmirGLO-HDMCP-3’UTR was used to transfect competent cell with CaCl₂. Thereafter, the transfected cells were cultured at 37℃for 16h, followed by pmirGLO-HDMCP-3’UTR extraction (TIANGEN, China) and verification by gene sequencing. Thirdly, the miR-146 mimics were synthesized and cotransfected both BRL-3A and L02 cells with pmirGLO-HDMCP-3’UTR using lipofectamin 2000 (Invitrogen, The USA). After culturing at 37℃/5% CO₂ for 25h, those cotransfected cells were harvested and the luciferase activity was tested using a dual-luciferase reporter gene detection kit (Promega, the USA).In this step, subjects were divided into four groups as followings: blank cell group, pmirGLO-HDMCP-3’UTR group, negative control miRNA+pmirGLO-HDMCP-3’UTR group and miR-146+pmirGLO- HDMCP-3’UTR group.

The wild-type and mutant 3’-UTR DNA fragments of SOCS5 covering the predicted binding sites of gga-miR-365-3p were successfully cloned. The psiCHECK™-2-SOCOS-3’UTR (wild type and mutant) vector was constructed by combining the luciferase vector psi-CHECK™-2 (Promega, Madison, WI, USA) with SOCOS 3’-UTR (wild type and mutant).
Dual-luciferase reporter assay, as we have reported before [19 (link)]. In a nutshell, when cells reached 80–90% confluence, using Lipofectamine 2000 (Invitrogen Life Technologies, USA) each co-transfected cells with wild-type or mutant reporter plasmid (200 ng) and 10 pmol of the indicated RNA oligonucleotides. Then, the luciferase activity in each group was detected using an automatic microplate reader (Bio-Rad, Hercules, CA, USA) in accordance with the dual luciferase reporter gene detection kit instructions (Promega, Madison, WI, USA) according to the manufacturer’s protocol.