microRNAs (miRNA) are abundant RNA molecules around 21 nucleotides long that regulate specific mRNA expression by directly interacting with the mRNA molecule. Our understanding of miRNA function in mRNA regulation has grown exponentially as more miRNA molecules have been described. As of 2013, more than 24,000 miRNA molecules had been described from more than 140 separate species, indicating that miRNA regulation is conserved across species. In humans, 2,500 mature miRNAs have been described, and researchers predict that 60% of human protein-coding genes may be targets of miRNA regulation. Most often miRNA regulation of an mRNA results in decreased expression, either by destabilizing the mRNA or by inducing translational repression. Very recently, some researchers have reported up regulation of mRNA through miRNA activity.
Since miRNA molecules are so abundant within cells and across species and their target sequences are found in so many protein-coding genes, understanding how miRNA regulation of mRNAs acts in concert with the many other levels of gene expression regulation becomes a complex, but fundamental, biological question.
To probe miRNA regulation of mRNA, the proper tools and experimental design are essential.
In her webinar, “Analyze microRNA activity in cells using luciferase reporters,” Trista Schagat presented considerations for each step in the process of reporter-based experiments to study miRNAs.
One of the tools highlighted in the webinar was the pmirGlo Vector. The pmirGLO Vector is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites downstream or 3´ of the firefly luciferase gene (luc2). Firefly luciferase is the primary reporter gene; reduced firefly luciferase expression indicates the binding of endogenous or introduced miRNAs to the cloned miRNA target sequence. This vector is based on Promega dual-luciferase technology, with firefly luciferase (luc2) used as the primary reporter to monitor mRNA regulation and Renilla luciferase (hRluc-neo) acting as a control reporter for normalization and selection.
The properties of the pmirGLO vector that make it particularly amenable to miRNA studies include its minimal reporter. When looking for down regulation by miRNA regulation, you are essentially looking at a stoichiometric reaction: two RNA molecules interacting with each other. If your reporter is driven by too strong a promoter, it might be difficult to see any decrease in reporter expression resulting from miRNA interaction.
Additionally, all reporter assays, particularly those involving transient transfection of cells, benefit from a control to normalize results to transfection efficiency. Typically your experimental reporter is normalized to a second control reporter. The pmirGLO vector contains a control reporter gene (hRLuc-neo) within the vector itself, automatically providing a critical internal experimental control.
Other elements that a vector should have to make it useful include a polyA adenyation signal for the production of stable reporter mRNA and a multiple cloning region that allows you flexibility in designing your cloning scheme to include your sequences of interest.
In addition to a vector with the appropriate components, DNA preparation, transfection optimization and treatment controls are all essential to designing a meaningful miRNA reporter experiment. The webinar goes into greater detail about all of these aspects.
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