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. 2011 Jun 15;6(6):e21215. doi: 10.1371/journal.pone.0021215

Figure 12. Luciferase reporter assay to determine the specific targeting and the tolerance of AmiRs to mutations.

Figure 12

A) Design of the CVB3 3′UTR mutants. Part of the CVB3 3′UTR harboring the targeting sites of AmiR-1 and AmiR-2 was presented. Two mutants were designed by substitution of nucleotides which are underlined. The numbers indicate the positions of the nts within the 3′UTR of CVB3. B) Schematic diagram of the pmir-GLO-CVB3-3′UTR reporter construct. The CVB3 3′UTR sequences (wt or mut) were inserted after the 3′ end of firefly luciferase gene in the pmir-GLO vector. C) Luciferase Assay of the targeting effect of pRNA-AmiR chimerics. HeLa cells were co-transfected with pRNA-AmiR chimerics and luciferase reporter constructs containing wt or mutated CVB3 3′UTR. The cells transfected only with a given reporter construct listed in B) above served as a control in each specific group. Firefly and Renilla luciferase activities were detected using Dual-Glo luciferase analysis system 48 h after transfection. The ratio of luciferase activities (firefly/Renilla) was calculated and normalized to the one transfected only with empty pmir-GLO vector.