Dual Luciferase Gene Reporter Assays to Study miRNA Function

Abstract

This chapter describes one of the most reliable quantitative assays to test the silencing of a possible target gene by a specific miRNA using a luciferase reporter gene. The experimental procedure first consists in cloning both the wild-type and mutated forms of the 3'UTR of the miRNA predicted mRNA target downstream of a firefly luciferase reporter. Next, each construct is co-transfected together with the miRNA into HeLa cells, and the reporter expression is monitored. Changes in luciferase levels will indicate whether or not a miRNA can bind to the UTR and regulate its expression.

MicroRNA-141 inhibits the self-renewal of glioblastoma stem cells via Jagged1

“…Dual luciferase plasmids of Jagged1 and its corresponding mutant were modified from a psiCHECK2 vector (Promega Corporation, Madison, WI, USA) and dual luciferase assay was undertaken as instructed by the protocol provided by Promega Corporation (20). All primers used are listed in Table I.…”

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

“…These assays rely on established cell lines to permit the high-throughput screening of the enhancer activity of hundreds of different sequence fragments (29)(30)(31)(32). Mammalian cell lines have been used to identify the first minimal tonicity-responsive enhancer (TonE), also known as an "osmoticresponse element" (ORE) (33)(34)(35).…”

Guidelines for the functional annotation of microRNAs using the Gene Ontology

“…4). Briefly, if binding of the miRNA to the mRNA is demonstrated followed by a reduction in mRNA levels, for example, by showing thatapplication of the miRNA to the target mRNA 3 ′ UTR fused to a luciferase reporter results in decreased reporter expression while application to a mutated form of the 3 ′ UTR has no effect (Clément et al 2015), the miRNA should be annotated to the molecular function (MF) term "mRNA binding involved in post-transcriptional gene silencing by miRNA" (GO:1903231). If binding is not demonstrated but there is evidence of a reduction in the levels of an mRNA in responseto an miRNA, forexample, anexperiment showing that application of a given miRNA results in decreased target mRNA levels as measured by qRT-PCR (e.g., see Maegdefessel et al 2012), then either the BP term "negative regulation of gene expression" (GO:0010629) should be used (if the mRNA is not a predicted target) or "gene silencing by miRNA" (GO:0035195, if the mRNA is a predicted target).…”

“…A validated binding target is an mRNA that has undergone experimental investigation to determine that the miRNA both binds to and regulates the expression of the mRNA. The most applicable evidence is a reporter assay, where the miRNA is combined with a reporter-fused 3 ′ UTR of the mRNA and altered levels of reporter expression are observed (Clément et al 2015). Also acceptable is an assay demonstrating physical interaction between the miRNA:mRNA, e.g., affinity purification, together with an assay demonstrating that the miRNA alters the levels of either the mRNA (e.g., qRT-PCR) or protein (e.g., Western blot) (Dangwal et al 2012).…”

“…Caution must be used by any curator wishing to infer knowledge about the function of an miRNA in one species from experimental data for an miRNA from another species. This is because even slight variations in the sequences of the mRNA or miRNA can cause loss of complementarity and disruption of the interaction, as demonstrated when using a mutated form of either the miRNA or mRNA in a standard reporter assay used to validate targets of miRNAs (Clément et al 2015). Individual annotation groups will have different policies with respect to whether or not annotations are transferred to miRNAs from other species.…”