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Efficient Detection and Analysis of miRNAs

Innovative Tools for Screening Hundreds of miRNAs and Subsequent Functional Studies

• Subu Yerramilli, Ph.D.
• ,
• Martin Kreutz
• ,
• Yexun Wang, Ph.D.
• ,
• Elizabeth Scanlan, Ph.D.

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miRNA Target Verification

Figure 3. Target verification: MCF-7 cells were co-transfected with an miR-29a mimic plus a nonbinding target protector as negative control or miR-29a plus a target protector specific for the putative miR-29a binding site of DNMT3A (mimic + target protector). After transfection, expression analysis of DNMT3A was performed by real-time RT-PCR with untransfected cells also being analyzed. Negative control: control siRNA and control target protector. Click Image To Enlarge +

Figure 3. Target verification: MCF-7 cells were co-transfected with an miR-29a mimic plus a nonbinding target protector as negative control or miR-29a plus a target protector specific for the putative miR-29a binding site of DNMT3A (mimic + target protector). After transfection, expression analysis of DNMT3A was performed by real-time RT-PCR with untransfected cells also being analyzed. Negative control: control siRNA and control target protector.

Target prediction algorithms allow identification of putative miRNA-binding sites for genes of interest (e.g., TargetScan). This can be verified in vitro using target protectors.

An example is the DNMT3A gene that encodes DNA methyltransferase 3, which is involved in development and differentiation. TargetScan predicted a single binding site for the miRNA miR-29a in the 3´ UTR of DNMT3A. Transfection of a miR-29a mimic led to downregulation of DNMT3A as shown by real-time RT-PCR, indicating that miR-29a downregulates DNMT3A expression at the transcriptional level.

Co-transfection of a target protector designed for the miR-29a binding site resulted in an increase in DNMT3A expression. These results provided convincing evidence that DNMT3A is negatively regulated by miR-29a via the binding site predicted by TargetScan (Figure 3).

Innovative tools such as the RT2 miRNA PCR arrays from Qiagen enable scientists to screen hundreds of miRNAs in one experiment, facilitating the understanding of the role of miRNAs in their area of interest. The experiment is simple to perform and provides sensitive, reproducible, and reliable results to accurately profile multiple genes simultaneously.

Verification of the results and in-depth functional analysis can then be undertaken by other techniques such as transfection with miRNA mimics and inhibitors and subsequent expression analysis.