Another method you could use do determine if a target is a direct target of a particular miRNA is with luciferase assays (see products such as Ambion's pMIR-REPORT system or promega's pmirGLO dual luciferase assays). Luciferase reporter constructs can be constructed with vectors bearing specific miRNA binding sites, by cloning the target sequence into a vector known as pMIR-REPORT, creating a wild type construct. A mutagenic version of the construct (negative control) will also be created by performing site-directed mutagenesis. Additional controls for this experiment can include a plasmid without a miRNA target region cloned in, reverse target orientation control and a part of the target region that doesn’t harbor the binding site of interest (Kuhn et al., 2009). Subsequently, a pMIR-REPORT luciferase assay can be conducted. The pMIR-REPORT (WT) construct and the specific miRNA will be co- transfected. If the miRNA interacts with the predicted miRNA target, a decrease in luciferase activity will be evident. Co-transfection with the negative controls should not result in lowered luciferase activity.

Thank you stefanie but I have performed the bioinformatic analysis as a bioinformatition and our wet lab partner is doing the RACE PCR to validate the targets so I need to know how much time does it take to run a PCR (as a curious cat :-) )