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

sing siPORT NeoFX Transfection Agent (Ambion). Reduction in endogenous enzyme activity or gene expression by each of the Pre-miR miRNAs was expressed relative to cells transfected with a negative control Pre-miR. Expression of G6PD (miR-1b) and Caspase 3 (let-7a) were measured using enzymatic assays specific to the two target proteins. Expression of HOXB8 was monitored using real-time PCR as miR-196 has been shown to cleave the HOXB8 mRNA [4].


miRNA Inhibitors

Two recent publications describe the use of antisense RNA molecules to inhibit the activities of specific miRNAs [5, 6]. The activities of the endogenous miRNAs were measured after co-transfecting cells with individual reporter constructs that included an miRNA target site as described [6]. High miRNA activity would result in lower reporter gene activity, and inhibiting miRNA activity would increase reporter gene activity. On average, the Anti-miR miRNA Inhibitors induced an approximately 4-fold increase in the expression of the reporter relative to cells co-transfected with a negative control inhibitor and the various miRNA reporter plasmids (Figure 4). Two negative control samples were included in this experiment to emphasize the specificity of these results: a luciferase reporter construct that does not contain any miRNA target sites and a luciferase reporter construct containing a target site for a brain specific miRNA not expressed in HeLa cells. Both had high luciferase activity as expected.

Figure 4. Enhanced Expression of miRNA-regulated Genes by Anti-miR Inhibitors. HeLa cells (5 x 104 cells/well) were plated in 24 well plates. Duplicate
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