3'UTR microRNA Target Clones

3'UTR microRNA target clones for luciferase reporter validation of predicted targets

When you have a computational miRNA target prediction and you need to test whether the miRNA actually represses that mRNA, 3'UTR target clones are the workhorse. The construct fuses a candidate 3'UTR sequence downstream of a luciferase reporter; co-transfecting with a miRNA mimic or expression construct gives you a quantitative reporter readout of binding-driven repression.

Work through the construct decision.

1. Reporter chemistry. For secreted-reporter kinetic time-course choose GLuc-based vectors. For internal normalisation choose dual-reporter (GLuc plus SEAP). 

2. Vector backbone. For transient assays in standard cell lines choose pEZX-MT05 or pEZX-MT06. For lentiviral integration into stable lines choose pEZX-LvPF02 variants.

3. Controls. Always include a paired mutant-seed control (same 3'UTR with the miRNA seed-binding site mutated). The mutant-seed control rules out non-specific reporter response.

4. Species. Human, mouse, and rat are standard.

5. Scale. For primary screens validate two to three top miRNA-target pairs before scaling to many. GeneCopoeia carries the catalogue in your inventory.