miR-132 Sponge Efficacy and Specificity.

<p>To test the efficacy of the sponge we examined whether it altered miR-132 expression in rat PC12 cells. <b>a,</b> Treatment of PC12 cells with NGF resulted in a 1.7 fold increase in mature miR-132 expression (p<.001, Newman-Keul test). Infection with the miR-132 sponge prevented this miR-132 induction. To determine the effect of miR-132 induction on a target, we measured mCherry expression in a monoclonal PC12 cell-line expressing the miR-132 inverse reporter. As expected, NGF treatment significantly reduced mCherry expression and this reduction was blocked by the miR-132 sponge. <b>b,</b> To test the specificity of the sponge we examined the microRNA expression profile of HEK293 cells infected with the miR-132 sponge. We selected HEK293 cells because they have a very low abundance of miR-132, thus limiting possible indirect effects on other microRNAs as a result of miR-132 knockdown. Using the TaqMan multiplex array we robustly detected 86 microRNAs (Ct<34). Of these, only seven were reduced by more than 25% in the sponge-expressing cells. We cross-referenced these seven microRNAs to microarray data examining microRNAs expressed in the adult dentate gyrus (Luikart, unpublished data). Only three microRNAs were both reduced in the multiplex array and expressed endogenously in the dentate gyrus – miR-187, miR-218, and miR-301. A single multiplex array can yield highly variable results. We therefore assessed whether these potential off-target effects could be validated using larger sample sizes (n = 3 independent experiments) and real-time PCR. <b>c,</b> Infection of PC12 cells with the miR-132 sponge resulted in a 32% reduction in miR-132 expression, but did not reduce the expression of miRs-187, 218, 301, or 212 (p>0.3; ANOVA with Tukey post-hoc). miR-212 is highly homologous to miR-132 and generated from the same transcript, making it a particularly good specificity control.