Syn mmu mir 181a miscript mirna mimic

Luciferase reporter assays were used to verify direct interactions between the miRNAs and the target genes. 3′-UTR region of Smad7, suppressor of cytokine signaling-3 (Socs3), and Tgfbr1 were PCR-amplified from mouse genomic DNA, using primers containing appropriate restriction sites and then cloned into psiCheck vector, a Renilla luciferase reporter vector (Promega). The following primer sequences were used to amplify specific 3′-UTR fragments.
Correct orientation and sequences were confirmed by restriction digestion and sequencing.
To assess the impact of the miRNAs, recombinant plasmid DNA (200 ng) and miRNA mimics (100 nM) were co-transfected into HEK293T cells using Attractene Transfection Reagent (Qiagen). Co-transfection with a scrambled miRNA sequence was used as a control. miR-181a and miR-181b mimics, negative control, and Attractene Transfection Reagent were purchased from Qiagen (Syn-mmu-miR-181a miScript miRNA Mimic, Syn-mmu-miR-181b miScript miRNA Mimic, AllStars Negative Control siRNA). Luciferase activity was measured using the Dual Luciferase system (Promega) 48 h post-transfection. Renilla luciferase levels were normalized to firefly luciferase activity as the internal transfection control.

Transfection assays were performed using Hiperfect Transfection Reagent according to manufacturer’s instructions (Qiagen). miRNA-181a and -b mimic as well as scrambled sequences were purchased from Qiagen (Syn-mmu-miR-181a miScript miRNA Mimic, Syn-mmu-miR-181b miScript miRNA Mimic, AllStars Negative Control siRNA). Briefly, 3 µl of Hiperfect Transfection Reagent was added to 100 µl of serum-free DMEM medium containing miR-181a or miR-181b mimics or negative control at a final concentration of 50 nM. The volume of cell culture medium in 24-well plates was adjusted to 600 µl with medium containing 10% FBS. To evaluate the transfection efficiency, we monitored green fluorescent protein (GFP) expression using fluorescence microscope. The percentage of GFP-positive cells varied between 60 and 65% for lymphocytes and 80 and 85% for HEK cells. We also measured miR181a and -b expression in transfected lymphocytes by real-time PCR (Figure S1 in Supplementary Material).