HeLa cells were transfected with a plasmid with a Renilla luciferase (transfection efficiency control) and miRNA target sequence in the 3’UTR of a firefly luciferase reporter gene. The next day, the cell cultures were transfected with different concentrations of the corresponding miRCURY LNA miRNA Mimics or cel-miR-39-3p negative control. Firefly luciferase expression is suppressed by the corresponding mimic at endogenous miRNA levels in the cell. Reporter gene expression was measured 24 H after transfection with a dual luciferase assay. 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 mimics display sub-nanomolar potency under optimal transfection conditions.
HeLa cells harboring hsa-miR-16-3p (top panel) and hsa-miR-16-5p (bottom panel) luciferase reporter plasmids, were transfected with hsa-miR-16-3p and hsa-miR-16-5p mimics, respectively, and Cel-miR-39-3p negative mimic control. The results demonstrate that suppression of luciferase actity is only achieved with the miRCURY LNA miRNA Mimic corresponding to the reporter assay.
Note that the fluorophore is attached to the 5’ passenger strand and not to the miRNA guide strand.
Introduction of fluorophore at the 5' end of the passenger strand has no adverse effects on the miRNA acitivity of the mimic. HeLa cells harboring an hsa-miR-1-3p luciferase reporter plasmid were transfected with fluorescence-labeled and non-labeled hsa-miR-1-3p mimics and Cel-miR-39-3p negative mimic control. The results demonstrate that suppression of luciferase actity is achieved equally efficiently with fluorescence-labeled and non-labeled mimics.
HeLa cells were transfected with a plasmid harboring a Renilla luciferase gene (transfection efficiency control) and a firefly luciferase reporter gene with a sequence complementary to hsa-miR- 1-3p in the 3' UTR (pmiR-1). The next day, the cell cultures were transfected with different concentrations of hsa-miR-1-3p miRNA mimic or Cel-miR-39-3p negative control. Reporter gene expression was measured 24 H after transfection with a dual luciferase assay. 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 potent miRNA activity can be achieved with biotinylated mimics even if they are ~50x less active than unlabeled mimics.
Biotinylated miRCURY LNA miRNA Mimics consist of three RNA strands: a 3’ biotinylated miRNA (guide) strand with sequence exactly according to miRBase annotation and the passenger strand, which is split into two LNA-modified RNA strands complementary to the miRNA strand. Only the miRNA strand is incorporated by the RISC. The two passenger strands are too short to act as miRNAs and are rapidly degraded after displacement from the miRNA strand.
miRCURY LNA miRNA Mimics consist of three RNA strands. An unmodified miRNA (guide) strand has a sequence exactly matching the miRBase annotation. The passenger strand is split into two LNA-modified RNA strands complementary to the miRNA strand.
Only the miRNA strand is incorporated by RISC. The two passenger strands are too short to act as miRNAs and are rapidly degraded after displacement from the miRNA strand. Off-target effects from the passenger strands are therefore minimal with miRCURY LNA miRNA Mimics.
Biotinylated miRNA mimics are delivered to cells by transfection. Cells are harvested and lysed 24 H later. Complexes of biotinylated RISC and mRNA are pulled down with streptavidin-coated magnetic beads. After washing, the captured RNA is purified and analyzed with microarrays, RT-qPCR or by next-generation sequencing.
