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Fig. 5 | BMC Genomics

Fig. 5

From: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

Fig. 5

miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b Validation of the MS2-mediated affinity purification strategy by RT-qPCR quantification of GFP transcripts with and without the MS2 RNA motif. Fold change was calculated using Pfaffl’s method [61]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p-values were calculated using the Mann Whitney test. Note scale bar differences between the graphs

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