The Next Step: Functional Studies
After miRNAs involved in a disease or pathway of interest have been identified by a PCR array, functional studies can provide further insights, such as identifying miRNA targets and binding sites as well as pinpointing the role of a particular miRNA in a pathway or process.
This type of investigation can be performed using strategies that involve transfection of functional molecules, such as synthetic miRNA mimics, miRNA inhibitors, or miRNA target protectors. A mimic behaves like a mature endogenous miRNA after its transfection, while miRNA inhibitors bind to an miRNA and inhibit its function.
Transfection of a mimic or inhibitor can be followed by phenotype or expression analysis. Target protectors allow the researcher to interfere with the interaction between a given miRNA and its specific target, leaving other targets of the miRNA unaffected. This is achieved because the target protector binds specifically to the miRNA-binding site in the 3´ UTR of an mRNA of one gene preventing miRNA binding and thereby preventing miRNA-mediated regulation (Figure 2).