Three prime untranslated region (3'UTR) reporter constructs are widely used by the scientific community to functionally link microRNAs (miRNAs) to suppression of mRNA expression. However, full-length 3'UTR vectors are rarely employed due to labor-intensive cloning work. Instead, 3'UTR fragments containing putative miRNA binding sites are commonly utilized to mechanistically validate miRNAs. Assaying truncated 3'UTRs may falsely validate miRNAs due to altered positioning of binding sites in respect to 3'UTR length and RNA secondary structure. Here we present a detailed protocol for the construction of full-length 3'UTR luciferase reporter constructs that was used to unveil miRNAs regulating multiple cell-cycle factors.
Keywords: 3′UTR; Dual-luciferase vector; G418 selection; Genomic DNA; Nested PCR; Restriction enzyme digestion; U2OS cells.