For shRNA reporter assays, gBlocks (Integrated DNA Technologies, Inc., IDT) with shRNA binding sites were inserted into pTtNPT or pRSF91.mTagBFP2.Sensor.WPRE as described62 to generate stable 32D reporter cell lines, which were then transduced with shRNA constructs to perform the reporter assay62.
The 3′UTR fragments of target genes containing wild-type (WT) or seed region mutated (Mu) of miR-188 binding sites were inserted into a dual-luciferase reporter vector.
The 3′UTR of wild-type NFAT5 and a variant containing mutations in the putative miR-568 binding sites were inserted downstream of the firefly luciferase gene in the pGL3 vector (Promega, Madison, WI, USA).
Binding sites may (a) duplicate, where a copy of a binding site is inserted in front of a random gene, (b) be deleted, (c) have their weight w changed from activating to inhibiting and vice versa, (d) have their binding specificity changed to a random other value.
To determine whether the negative regulatory effects of miR-10a on BCL6 expression were mediated by the binding of miR-10a to the predicted target sites in the 3′-UTR of the BCL6 mRNA, the full-length 3′-UTR of BCL6 containing the predicted miR-10a binding site was inserted downstream of the firefly luciferase gene in a reporter plasmid.
The mini-chromosome pARS1/-B23/G24 (Fig. 1B) carries ARS1, in which two of the auxiliary elements, B2 and B3, are destroyed and a GAL4 binding site is inserted instead of B3 [20].
The 3′UTR of the wild-type IGF1R and a variant containing mutations in the putative miR-375 binding site were inserted downstream of the firefly luciferase gene in the pGL3 vector (Promega, Madison, USA).
To construct luciferase reporter vectors, oligonucleotides (Biomers) comprising the wildtype or mutated binding site were inserted downstream of the Firefly luciferase gene into the pmir-GLO Dual-Luciferase miRNA Target Expression Vector (Promega) according to the manufacturer's instructions.
A 1,140 bp region of the 3'UTR of MAP3K9 containing the predicted miR-34a binding site was inserted into the dual luciferase PsiCheck2 reporter vector (Promega), designated Psi/miR-34a (Additional File 2, Figure S2a).
To determine whether miR-146 could directly repress EGR-3 we performed luciferase assays in which a region of the EGR-3 3′ UTR or a concatemer of the predicted miR-146 binding site, were inserted downstream of the luciferase open reading frame (ORF).
