This plate shows the layout of panel 10 of the human miRNA inhibitor library.
HeLa cells were transfected with a plasmid containing Renilla luciferase for the transfection efficiency control and the miRNA target sequence of a firefly luciferase reporter gene in the 3’ UTR. Firefly luciferase expression is suppressed by the corresponding endogenous miRNA level in the cell. One day later, the cell cultures were transfected with various concentrations of the corresponding regular miRCURY LNA miRNA Inhibitor. Reporter gene expression was measured with a dual luciferase assay 24 H after transfection. Ratios of firefly and Renilla luciferase activity were calculated and normalized to values obtained with a firefly luciferase reporter with no miR target sequence (pLuc). The results illustrate that our regular miRCURY LNA miRNA Inhibitors display sub-nanomolar potency under optimal transfection conditions.
HepG2, HeLa and HEK293 cells were transfected with a plasmid encoding Renilla luciferase (transfection efficiency control) and a firefly luciferase reporter gene with either an miR-21-5p target site or an miR-27a-3p target site in the 3’-UTR (pmiR-21-5p or pmiR-27a-3p). 24 H after removal of the transfection reagent, corresponding miRCURY LNA miRNA Power Inhibitors were added directly to culture medium in different concentrations. Reporter gene expression was measured with a dual luciferase assay 48 H (HepG2) and 72 H (HeLa and HEK293) after addition of the inhibitors. Ratios of firefly and Renilla luciferase activity were calculated and normalized to values obtained with a firefly luciferase reporter with no miR target sequence (pLuc) in each of the three cell lines.
The results illustrate that efficient miRNA inhibition can be achieved by adding high concentrations of Power Inhibitor directly to the culture medium. However, uptake kinetics with gymnosis is slower than delivery of the inhibitors using transfection reagents. When using transfection reagents, we normally observe strong inhibition after just 24 H. With unassisted uptake, we observe activity with some cell lines and certain inhibitors after one day, but it typically peaks between 48–72 H after addition of the inhibitors. Normal inhibitors with an unmodified, normal phosphodiester backbone are ineffective with gymnotic delivery, probably due to insufficient stability.