Pmir report β gal

Using four computer algorithms, including TargetScan, miRanda, PicTar and miRGen, we identified PTN as a possible miR‐384 target. The sites were predicted based on the base‐pairings of seed‐sequence matches. To construct reporter plasmids containing wild‐type or mutant miR‐384 target sites for human PTN 3′UTR segments, oligonucleotide pairs containing the desired miR‐384 target region and restriction enzymes sites were annealed and ligated into the pMIR‐REPORT™ miRNA Expression Reporter Vector (Ambion, Grand Island NY, USA). The mutated putative miR‐384 binding site in the PTN 3′UTR was generated using a QuikChange site‐directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol. These cells were plated in 12‐well plates (3 × 10⁴ cells/well). Twenty‐four hours later, the pMIR‐PTN‐3′‐UTR construct (200 ng) and the β‐gal expression vector pMIR‐REPORT‐β‐gal (200 ng) (Ambion) were cotransfected with a miR‐384 mimic or a mimic control at a final concentration of 100 nM using Transfast™ Transfection Reagent (Promega). Twenty‐four hours after transfection, cell lysates were collected, and luciferase activity was measured according to the manufacturer's protocol. Firefly luciferase activity was normalized to β‐gal expression for each sample.