Guidelines for transfection

Guidelines for transfection of miRNA

microRNAs (miRNAs) are a class of endogenous small RNA molecules with similar characteristics to siRNAs. In recent years, it has been discovered that miRNAs play a role in many diverse biological processes such as development, differentiation, and apoptosis. Misregulation of miRNA expression is reported to be associated with several cancers and other diseases.

The miRNA system is an endogenous mechanism of regulation of gene expression. Mature miRNAs contribute to the regulation of endogenous genes, primarily by translational repression. In addition, miRNAs can mediate mRNA destruction by rapid deadenylation and/or decapping. Naturally occurring miRNA-binding sites are typically found in the 3' untranslated regions (UTRs) of target mRNAs. Their partial complementarity has made positive identification of true binding sites difficult and imprecise.

Transfection of miRNA mimics or inhibitors is a technique used to identify the targets and roles of particular miRNAs. miRNA mimics are chemically synthesized miRNAs which mimic naturally occurring miRNAs after transfection into the cell. miRNA inhibitors are single-stranded, modified RNAs which, after transfection, specifically inhibit miRNA function. Reduced gene expression after transfection of an miRNA mimic or increased expression after transfection of an miRNA inhibitor provides evidence that the miRNA under study is involved in regulation of that gene. Alternatively, the role of miRNAs in various pathways can be studied by examination of a specific phenotype following miRNA mimic or inhibitor transfection.

When using 24-well plates, we recommend that cells are seeded in wells first, followed by addition of mimic/inhibitor–reagent complexes in order to ensure optimal mixing of cells and complexes. However, reverse transfection, where complexes are added to wells first and then cells are added on top of complexes, can be performed if desired. To perform a reverse transfection, simply change the order in which cells and complexes are added to the plate.

However, for 96-well plates, we recommend using reverse transfection, since reverse transfection is rapid and convenient, and is frequently used for high-throughput formats. Reverse transfection is also optimal for cotransfection of miRNA mimic and miRNA inhibitor in 24-well plates.

Many miRNA experiments involve cotransfection of an miRNA mimic and/or inhibitor together with a plasmid DNA vector in which miRNA-binding sites are fused to a reporter gene, such as luciferase.

The amount of miRNA mimic/inhibitor needed to efficiently downregulate a target gene or inhibit miRNA function can vary greatly, depending on the miRNA, the cell line, and the chosen analysis method. To determine the concentration that provides optimal results, optimization experiments using varying mimic/inhibitor concentrations should be performed.

miRNA mimics can inhibit target protein expression at a final concentration as low as 0.5 nM. However, a higher concentration may be required, especially if performing downstream analysis at the protein level. miRNA inhibitors have been shown to inhibit miRNA function at a concentration of 50 nM. Lower inhibitor concentrations may also be effective.

In addition to optimization of concentration, time-course experiments may also be necessary to determine the optimal time after transfection for analysis of results. miRNA mimic or inhibitor effects often do not lead to an immediate change in transcript or protein levels.

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