<i>In vitro</i> target assay for <i>microRNAs</i> on the <i>CDK3</i> transcript.

<p>[A] Diagram of <i>miR-1744</i> binding sites in the 3′-UTR of the <i>CDK3</i>gene. [B] Expression vector maps for eGFP within the 3′-UTR of the <i>CDK3</i> gene and Ds-Red within each miRNA. The 3′-UTR of the <i>CDK3</i> transcript was subcloned between the <i>eGFP</i> gene and the polyA tail to generate the fusion construct for the GFP transcript following the miRNA target 3′-UTR (pcDNA-eGFP-3′UTR) (upper panel) and the miRNA expression vector was designed to co-express DsRed and each miRNA (pcDNA-DsRed-miRNA) (lower panel). [C and D] After co-transfection of pcDNA-eGFP-3′UTR for the <i>CDK3</i> transcript and pcDNA-DsRed-miRNA for the <i>miR-1744,</i> the fluorescence signals of GFP and DsRed were detected using FACS [C] and fluorescent microscopy [D]. See <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051592#s4" target="_blank">Materials and Methods</a></i> for complete description.