High fidelity enzyme

Potential miR‐196a‐5p binding sites of NR6A1 3′‐UTR were predicted by Targetscan (http://www.targetscan.org/). The fragment of NR6A1‐3′‐UTR‐wild‐type (NR6A1‐3′‐UTR‐wt) with miR‐196a‐5p binding sites and its mutant (NR6A1‐3′‐UTR‐mut) were digested with restriction enzyme Xho I and Not I, then cloned into pmiR‐RB‐REPORT plasmids (Guangzhou RiboBio), respectively. Then the recombinant plasmids and miR‐196a‐5p mimics/NC mimics were co‐transfected into HEK293T cells using TurboFect^TM. Relative Renilla activities were measured 48 h after transfection and firefly luciferase activity was normalized by luciferase activity.
The different truncations of CDH1 promoter containing three DR0 sites were PCR amplified by high fidelity enzyme (Transgen biotech) using human genomic DNA as template, then, respectively, cloned into pGL3 basic reporter plasmids (Promega) by double digestion of Kpn I and Hind III. All recombinant plasmids were sequenced and have no mutation. Recombinant naming was based on the positions of the promoter fragments. The recombinant plasmids contained −1938 to −1927 (pGL3 CDH1‐a), −912 to −917(pGL3 CDH1‐b), −167 to −178 (pGL3 CDH1‐c). All the recombinant plasmids were mixed with Renilla vectors, then co‐transfected into HEK293T cells with NR6A1. The relative fluorescence value is calculated by ratio of Luc/Ren.