title: Design and interpretation of microRNA-reporter gene activity.
abstract: MicroRNAs (miRNAs) are small noncoding RNA molecules that act as sequence specificity guides to direct post-transcriptional gene silencing. In doing so, miRNAs regulate many critical developmental processes, including cellular proliferation, differentiation, migration, and apoptosis, as well as more specialized biological functions such as dendritic spine development and synaptogenesis. Interactions between miRNAs and their miRNA recognition elements occur via partial complementarity, rendering tremendous redundancy in targeting such that miRNAs are predicted to regulate 60% of the genome, with each miRNA estimated to regulate more than 200 genes. Because these predictions are prone to false positives and false negatives, there is an ever present need to provide material support to these assertions to firmly establish the biological function of specific miRNAs in both normal and pathophysiological contexts. Using schizophrenia-associated miR-181b as an example, we present detailed guidelines and novel insights for the rapid establishment of a streamlined miRNA-reporter gene assay and explore various design concepts for miRNA-reporter gene applications, including bidirectional miRNA modulation. In exemplifying this approach, we report seven novel miR-181b target sites for five schizophrenia candidate genes (DISC1, BDNF, ENKUR, GRIA1, and GRIK1) and dissect a number of vital concepts regarding future developments for miRNA-reporter gene assays and the interpretation of their results.
authors.affiliation: School of Biomedical Sciences and Pharmacy, Faculty of Health and Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Carroll
authors.forename: Paul A
authors.initials: PA
authors.lastname: Tooney
authors.forename: Murray J
authors.initials: MJ
authors.lastname: Cairns
mesh_terms: D005188:False Negative Reactions; D005189:False Positive Reactions; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*
chemical_list: C540193:MIrn181 microRNA, human; D035683:MicroRNAs