To better understand miRNA contributions to normal and diseased cell physiology, Thermo Fisher Scientific has developed what it says is a unique approach for expressing mature miRNAs. According to Annaleen Vermeulen, Ph.D., senior scientist, there are several approaches for functionally analyzing miRNAs.
“While synthetic miRNA mimics work well for transient overexpression in vitro, some cells are resistant to the delivery methods required, such as lipid transfection. Lentiviral vectors can overcome this limitation, but expression presents other challenges. Existing approaches rely on expression of the native primary miRNA, which is subject to innate regulation by the cell and can yield ambiguous results across cell types and conditions.”
To address the issues posed by inconsistent processing of expressed miRNA, the company developed a novel design strategy. “Our latest miRNA technology, the miRIDIAN® shMIMIC™ Lentiviral miRNA, embeds each human mature miRNA annotated in the miRBase into a highly optimized, universal primary miRNA scaffold. As such, we are able to maintain critical secondary structural elements for the consistent, reproducible processing of mature miRNA.”
Alex Amiet, senior product manager, RNAi, explained that “each shMIMIC miRNA has been designed, cloned, and packaged into lentiviral particles so the researcher can transduce into biologically relevant cells, including difficult-to-transfect primary, neuronal, and stem cells.
“The optimized, universal primary miRNA scaffold ensures robust enzymatic processing and transport of miRNA in a consistent manner within any given cell. Additionally, because the lentiviral vector allows integration of the shMIMIC miRNA into the host cell genome, researchers can create stable cell lines.”
Amiet noted that investigators still need to optimize certain parameters such as cell number and viral transduction conditions. “Overall, it is fairly straightforward, since we provide concentrated lentiviral particles. For the future, we may expand the collection to include mouse and rat miRNAs. At present, since miRNAs are evolutionarily conserved among mammals, many human shMIMIC miRNAs may be applicable in rodent models.”