Figure 3. UTA Reporter derived functions uncouple miRNA expression from functionality.

(A) Expression levels of miRNAs were investigated by NGS using three small-RNA libraries from three independent HEK293 cultures. Sequence data were processed and aligned to sRNA databases, subsequently reads were normalized using DEseq package. Sample quality and sequencing reproducibility were assured by using heat map and blind hierarchal clustering. Selected miRNA target regions were inserted into the CFP 3′UTR, transfected into HEK293 cells and incubated for 72 h, then cells were examined by FACS and transfer functions were calculated (for details see methods section). Three different functional behaviors are depicted according to the shape of the transfer function: (B) High functional (Left) have a threshold up to high YFP intensities, (C) mid Functional (Middle) have a threshold at intermediate YFP intensities and (D) low functional (Right) have no detectable threshold (compare Supplementary Fig. S3). (E) Functional groups were used as ordinal variables and mean normalized counts were compared between groups (P < 0.01 Kruskal-Wallis test, Dunns multiple comparisons test * < 0.05).