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Reagents for Up and Down-Regulation of miRNA Activity in Mammalian Cells

molecules modeled after miRNA precursors could enter the miRNA pathway and regulate translation. Using an miRNA target containing a luciferase reporter to monitor miRNA activity, we tested three different miRNA precursor designs for five specific miRNAs. All three synthetic precursor miRNA designs resulted in decreased reporter activity, with Design #3 providing the strongest miRNA activity (i.e. inhibition of a target reporter construct) (Figure 1). Based on these and other similar experiments, Design #3 was used to develop Ambion's Pre-miR miRNAs.

Figure 1. Comparison of Synthetic Precursor miRNA Designs. In 24 well plates, ~40,000 HeLa cells were co-transfected with luciferase reporter constructs bearing targets sites for either miR-10, miR-33, miR-124, miR-19, or miR-130, and 10 nM of either a negative control precursor miRNA (not shown) or one of three different precursor miRNA designs for the appropriate miRNA (#1, #2, #3). The cells were assayed for luciferase activity 48 hours post-transfection. The reduction in luciferase activity in the Pre-miR miRNA-transfected cells was measured relative to the luciferase activity in the negative control-transfected cells. The data are averages of triplicate transfections.


We also wanted to determine whether Pre-miR miRNAs can regulate endogenous genes whose expression patterns are known to be modulated by miRNAs. Design #3 was used to create synthetic precursors of miR-1b, let-7a, and miR-196, known to regulate expression of G6PD [3], Caspase 3 [2], and HOXB8 [4], respectively. To find an informative cell model, we used an miRNA array protocol (see

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