Figure 10

MiR-281 target study. (A) RNAhybrid prediction for miR-281 target sites and putative target sequences in the 5′-UTR of DENV-2 gRNA. (B) Cloning strategy of the 5′-UTR and mutant 5′-UTR upstream of the EGFP reporter gene ORF and downstream of pIB in the pIB/V5-His vector, denoted as target-5UTR and mut-5UTR. Target sequences are underlined, nucleotides complementary to the miR-281 seed region are shown in bold italic type. (C) Constructs were co-transfected with the miR-281 mimic or control mimic into C6/36 cells. Analysis by RT-qPCR shows that expression of EGFP was significantly higher in target-5UTR and miR-281 co-transfected cells than in mut-5UTR and miR-281 co-transfected as well as target-5UTR and control mimic-transfected cells. The RT-qPCR data were analyzed normalized to the blasticidin resistance gene. The RT-qPCR data were analyzed using the 2^-Δct method. Data shown represent three independent experiments with three triplicates each. P values are from ANOVA.