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. 2014 Mar 5;42(9):6064–6077. doi: 10.1093/nar/gku171

Figure 2.

Figure 2.

Rationally designed artificial miRNAs significantly repress PC and GLS 3′ UTR luciferase reporter genes. (A) Artificial miRNAs with seed matches targeting PC and GLS 3′ UTRs (blue) or non-targeting control miRNAs without seed matches (orange) were screened for repression of PC, GLS and GAPDH 3′ UTR luciferase reporter activity or empty vector. Bars represent the mean ± SD of reporter activity for triplicate transfections relative to the reporter alone control. Wilcoxon rank-sum tests were performed for each 3′ UTR to compare reporter activity with targeting miRNAs to activity with non-targeting controls, and only PC and GLS yielded P < 0.05, as indicated. (B) Relative GAPDH 3′ UTR reporter activity as in (A), but bars for miRNAs with seed matches to the GAPDH 3′ UTR are blue, while miRNAs lacking seed matches are orange. (C) Artificial miRNAs from (A) that repressed relative reporter activity > 2 SD below the mean of the reporter alone control and had a q < 0.05 (t-test versus the reporter alone control). Bars are coloured by whether the miRNA has a seed match to the indicated 3′ UTR reporter (blue) or lacks a seed match (orange). Bars representing the reporter alone control are red. Vec is the empty vector control. (D) Artificial miRNAs from (A) that significantly repressed both PC and GLS reporter activity. In each column, bars represent the activity of the indicated reporter for a single miRNA. Artificial miRNAs along the x-axis are sorted by the minimum relative activity across reporters for each miRNA. Bars are coloured by whether the miRNA was designed to target PC and GLS (blue) or was a non-targeting control (orange). Dark shading indicates the miRNA significantly repressed the corresponding reporter, while light shading indicates the reporter was not repressed. Bars representing the reporter alone control are red. (E) Artificial miRNAs that repressed both PC and GLS in the initial screen were re-assayed, and PC and GLS reporter activity for those miRNAs that validated is shown. Bars represent the mean ± SD of reporter activity for triplicate transfections of each miRNA (blue) relative to a commercially available non-targeting control (red). Bars in a column represent PC and GLS reporter activity for a single artificial miRNA, and miRNAs are sorted by the minimum relative activity across both reporters for each miRNA.