<i>In vitro</i> target assay of microRNAs on the SERPINB3 transcript.
2013-02-19T19:56:29Z (GMT) by
<p>[A] Diagram of <i>miR-101, miR-1668</i> and <i>miR-1681</i> binding sites in <i>SERPINB3</i> 3′-UTR. [B] Expression vector maps for eGFP with <i>SERPINB3</i> 3′-UTR and mutated <i>SERPINB3</i> 3′-UTR and Ds-Red with each miRNA. The wild-type (WT) and mutants of 3′-UTR of the <i>SERPINB3</i> transcript were subcloned between the eGFP gene and the polyA tail to generate the fusion construct of the GFP transcript following the miRNA target 3′-UTR (pcDNA-eGFP-3′UTR) (top and middle panel) and the miRNA expression vector was designed to co-express DsRed and each miRNA (pcDNA-DsRed-miRNA) (bottom panel). [C] After co-transfection of pcDNA-eGFP-3′UTR for the <i>SERPINB3</i> transcript and pcDNA-DsRed-miRNA for the <i>miR-101, miR-1668</i> and <i>miR-1681</i>, the fluorescence signals of GFP and DsRed were detected using fluorescent microscopy. [D] The rate of inhibition of eGFP expression from miRNA modulation was calculated by fluorescence-activated cell sorting (FACS). Solid bars represent WT of <i>SERPINB3</i> 3′-UTR and empty bars show the mutant of <i>SERPINB3</i> 3′-UTR for each miRNA. Error bars indicate the standard error of triplicate analyses. The asterisks denote statistically significant differences between WT vs. mutants (***P<0.001).</p>