title: 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.
authors.affiliation: Molecular Medicine Group, Robarts Research Institute, Western University, London, ON N6A 5B7, Canada. dmaribel@uwo.ca.
authors.forename: Danae
authors.initials: D
authors.lastname: Campos-Melo
authors.affiliation: Molecular Medicine Group, Robarts Research Institute, Western University, London, ON N6A 5B7, Canada. cdroppel@uwo.ca.
authors.forename: Cristian A
authors.initials: CA
authors.lastname: Droppelmann
authors.affiliation: Molecular Medicine Group, Robarts Research Institute, Western University, London, ON N6A 5B7, Canada. kvolkening@robarts.ca.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Volkening
authors.affiliation: Molecular Medicine Group, Robarts Research Institute, Western University, London, ON N6A 5B7, Canada. michael.strong@schulich.uwo.ca.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Strong
mesh_terms: D020413:3' Untranslated Regions; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods* / Q000592:standards; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D012680:Sensitivity and Specificity; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; D016900:Neurofilament Proteins; C044882:neurofilament protein L; D008156:Luciferases