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Figure 1 | BMC Genomics

Figure 1

From: Experimental evaluation does not reveal a direct effect of microRNA from the callipyge locus on DLK1 expression

Figure 1

Experimental evaluation of the effect of microRNA from the callipyge locus on DLK1 expression. (A) Overview of experimental procedure of ovine DLK1 reporter assay. We co-transfected COS1 cells with a mixture of ovine full-length (5’ UTR, ORF, and 3’ UTR) DLK1-expressing vector (p3.1M-DLK1-HA), GFP-expressing vector (pcDNA3-GFP) for normalizing transfection efficiency and gel-loaded protein amount, and one of 121 mimic miRNA candidates from the DLK1-GTL2 imprinted domain, two negative controls (NC1, NC2), and one siRNA targeting the DLK1 ORF as positive control. To facilitate protein quantification, an HA epitope was engineered at the carboxyterminal end of DLK1. Amounts of DLK1, GFP, and endogenous Tubulin were estimated by dual fluorescent Western blot (WB). Effect of each miRNA on DLK1 translation was estimated by normalizing DLK1 with either GFP or Tubulin amounts (corrected DLK1 amount). (B) Distribution of effects of the 121 mimic miRNAs on DLK1 protein amounts. Effects are expressed as fold difference with respect to the overall (minus siRNA) mean. The effects of two negative (NC1, NC2) and one positive (siRNA) controls are shown, as are the effects of the most inhibiting miR-329a-3p, the closely related miR-329b-3p, and the most activating miR-376a-5p and miR-432-3p. GFP was used for normalization. (C) Evaluation of synergistic action of multiple miRNA on DLK1 amount. We selected seven miRNAs consistently showing inhibiting, activating, and neutral effects on DLK1 amount. COS1 cells were co-transfected with the p3.1M-DLK1-HA and pcDNA3-GFP vectors along with either a mixture of the seven miRNAs (mix), or a single miRNA having shown the lowest (1st) or highest (last) corrected DLK1 amount upon transfection in each group. DLK1-HA and GFP protein levels were estimated by dual fluorescent WB analysis (upper panel). Means of corrected DLK1 amounts with standard deviations for four independent experiments are shown (lower panel).

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