As with any in vitro assay, PASSPORT-seq has some limitations. First, it detects only changes in mRNA, rather than protein levels. miRNA- mRNA interactions are known to cause mRNA destabilization, but can also lead to translational repression (Lim et al., 2005; Bartel, 2009). Since this assay does not detect changes in translation, it may underestimate the functional impact of some of the mirSNPs. Second, the variants could be affecting mRNA stability by mechanisms other than by altering miRNA targeting. For example, it could be altering RNA binding protein function that could alter the mRNA stability. Although this would need additional validation experiments to determine the mechanism of action, it would still be of biological value. Last, like other studies using the pISO plasmid, the miRNA binding site is tested in the context of the luciferase mRNA, rather than the endogenous mRNA.
