Comprehensive luciferase-based reporter gene assay reveals previously masked up-regulatory effects of miRNAs

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the majority of the transcriptome at a post-transcriptional level. Because of this critical role, it is important to ensure that the assays used to determine their functionality are robust and reproducible. Typically, the reporter gene assay in cell-based systems has been the first-line method to study miRNA functionality. In order to overcome some of the potential errors in interpretation that can be associated with this assay, we have developed a detailed protocol for the luciferase reporter gene assay that has been modified for miRNAs. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3'UTR (untranslated region) target. Our findings suggest that there is a real possibility that the roles for miRNA in transcriptome regulation may be misreported due to inaccurate normalization of experimental data and also that up-regulatory effects of miRNAs are not uncommon in cells. We propose to establish this comprehensive method as standard for miRNA luciferase reporter assays to avoid errors and misinterpretations in the functionality of miRNAs.

Figure 1

miRNA recognition elements (MREs) for miR-507 and miR-518e* within Firefly luciferase linked to the neurofilament (NEFL) mRNA 3'UTR.

Figure 2

Luciferase reporter assay using miR-507, miR-518e* or miR-let-7a and the NEFL 3'UTR. Data from reporter assays before (gray) and after the normalization (red) against the effect of the miRNAs on the Firefly luciferase reporter alone are shown. Data from Table 1 are expressed as positive values for up-regulation and negative values for down-regulation (X-1). Experiments were performed in triplicate. Results are shown as mean ± SEM. t-test was performed to compare the effect of each miRNA on the NEFL 3'UTR with the effect on the Firefly luciferase (*** = p < 0.001; n.s. = not significant). Comparison between miR-507 or miR-518e* with miR-let-7a negative control was performed using ANOVA, Newman-Keuls test (*** = p < 0.001).

Figure 3

Cellular and exogenous miRNA effects regulating the luciferase activity in the reporter gene assay. (a,b) Cells transfected with luciferase with the NEFL mRNA 3'UTR; (c,d) Cells transfected with luciferase without 3'UTR.