miRNAs regulate gene expression by either degrading the target mRNAs or by binding to mRNAs and blocking translation. Traditional luciferase assays test the effect of miRNA by measuring differences in its target protein activity. However, the differences in protein activity due to mRNA degradation and those from translational blockage will be indistinguishable using luciferase activity assays. The PASSPORT-seq assay provides additional evidence of the mechanism underlying the effect of the variant by specifically detecting only the changes in mRNA transcript levels. We demonstrated that the results obtained with our PASSPORT-seq assay did reflect those obtained with the traditional luciferase reporter assay set-up. As described above, the PASSPORT-seq assay quantifies the relative expression of luciferase mRNAs, whereas the luciferase assay measures the luciferase enzyme activity. Thus, it is not surprising that there may be differences in the magnitude of effects between the different assays. For example, the effect on the luciferase activity could be larger due to both the degradation of the mRNA and the blockade of translation. In contrast, the effect on the protein levels and activity could be smaller
