Focusing on miRNA Biogenesis
miRNA-profiling experiments can give a high-level overview of miRNA expression status under defined conditions. Following these experiments, researchers may wish to proceed by selecting smaller numbers of miRNAs with interesting expression profiles for further study of biogenesis and function.
Quantification of miRNA precursors alongside mature miRNAs can provide important insights into the pathways from transcription to maturation. Real-time PCR can be used to perform this type of analysis (e.g., using the miScript PCR System with assays specific for either mature or precursor miRNA).
Comparison of mature and precursor miRNA levels can reveal variations in relative levels of mature and precursor miRNA for different miRNAs, or mechanisms whereby mature miRNAs are processed from different precursor miRNAs under different conditions.
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).