Taq buffer

The 3’ untranslated regions (UTRs) of the gene sequences targeted by miRNAs were identified by Miranda (http://www.microrna.org/microrna/home.do) [24 (link)] and used to design oligonucleotides for the luciferase assay. The mRNA-3’ UTR region fragments were amplified using PCR. The fragment synthesis solution contained 10× Taq buffer (Solgent Co.), 10 mM dNTP mix (Solgent Co.), and h-taq (Solgent Co.). The synthesis conditions were an initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and a pause at 4°C. Then, the PCR product was purified using a PCR purification kit (Bioneer, Daejeon, Korea). The product was inserted into multiple cloning sites of a pmirGLO vector (Promega, Madison, WI, USA) that was linearized by a restriction enzyme. The linearized pmirGLO vector was mixed with the PCR product and ligase using a ligation protocol. The complete pmirGLO vector was transformed into competent cells and spread onto an ampicillin-containing agar plate. After 24 hours of incubation, colonies were selected and cultured in ampicillin-containing broth. Plasmid DNA was extracted using a plasmid mini extraction kit (Bioneer). The fidelity of the plasmid DNA sequence was confirmed by DNA sequencing.