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. 2017 Mar 24;7:45197. doi: 10.1038/srep45197

Figure 1. UTA Dual Fluorescence Reporter System functionally characterized miRNA activity.

Figure 1

Unstranslated Trans Assay (UTA) uses two independent fluorescent proteins expressed individually from two different promoters. RFP in A (or CFP in B) contained a perfect complementary target region for miRNAs within its 3′ UTR while YFP (or GFP) is unaffected (cartoons depict the used promoters and proteins). Human HEK293 cells were transfected with three different sensor constructs (miR-23a-3p, miR-27a-3p and non-cognate as control) and evaluated after 72 h. (A) Qualitative micrographs and (B) scatter plots for flow cytometry measurements display constant GFP/YFP expression in all the samples within a broad range of fluorescence intensities. The RFP/CFP expression was reduced for miRNA-23a-3p and miRNA-27a-3p targets but remained proportional to GFP/YFP in the non-cognate control. (C) Decreased ratios CFP/YFP for miRNA-targeted constructs from bulk quantitative analysis was performed after flow cytometry. Fluorescence intensity ratios of CFP and YFP for miRNA-23a-3p, miRNA-27a-3p and the non-cognate control were calculated using custom R scripts. (D) For standard luciferase assays HEK293 were transfected with miRNA-23a-3p and miRNA-27a-3p targets located within the 3′UTR of Renilla luciferase and the ratio between Renilla and Firefly luciferase were examined after 72 h.