Pmirglo dual luciferase mirna target expression vector pmirglo

The 3′un-translated region (UTR) of four predicted target genes for the identified miRNAs, TRIB3, M-SAA3.2, PTHLH, and VEGFA, were PCR amplified using DNA collected from the bovine mammary gland samples applied for sequencing in this study as a template, and connected into pmirGLO Dual-Luciferase miRNA Target Expression Vector (pmirGLO, Promega) (Figure 1), respectively. The primers were listed in the Table 1. Afterward, the connected products were transfected into Escherichia coli, and then verified the correct sequence and orientation by sequencing. The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, United States) was used to generate the 3′UTR variants of TRIB3, M-SAA3.2, PTHLH, and VEGFA where seed sequences recognized by microRNAs were deleted (Figures 2, 3). After the point mutation, same way was applied in order to find the correct mutant sequences for such four genes.

HEK293 cells were plated at 25% confluence 24 h prior to transfection. A cotransfection of a pmirGLO Dual-Luciferase miRNA target expression vector (pmirGLO) (Promega, USA) with SOCS3 or DUSP6 WT 3′ UTR or MT 3′ UTR (Supplementary Table 3), together with LNA-hsa-miRNA-181a (miRCURY) was performed in a 1:6 ratio with FuGENE6 (Promega, USA) according to the manufacturer’s protocol. Twenty-forty hours post transfection luciferase and Renilla luminescence were measured sequentially on a flow cytometric Discover microplate reader (Promega, USA).

The pmirGLO Dual-Luciferase miRNA Target Expression Vector (pmirGLO) (Promega, Madison, WI, USA) was used for the luciferase reporter assay. Briefly, two plasmid constructs, the IL6R wild type and IL6R mutated type, were created and cotransfected in cells using the Lipofectamine 3000 reagent (Thermo Fisher Scientific) along with different concentrations of the miR-125b-5p mimic (0, 5, 10, 25, and 50 µM) using the HiPerFect transfection reagent (Qiagen). The Dual-Glo® Luciferase Assay System (Promega) was used to measure the luciferase activity.