Guidelines for transfection
Performing appropriate miRNA control experimentsThe use of appropriate controls is essential for the correct interpretation of results from experiments using miRNA mimics or inhibitors. Every experiment should include a suitable positive and negative control. Additional controls may also be necessary to enable interpretation of results or troubleshooting.
miRNA mimic experiments — positive control
miRNA mimic experiments — negative control
miRNA inhibitor experiments — positive control
In experiments involving transfection of miRNA inhibitors, detection of the inhibitor effect is often complicated by the presence of other miRNAs in the cell which interact with the same target gene. Cotransfection of mimic and inhibitor should result in an increase in expression when compared to the mimic alone. This confirms that the inhibitor is effectively inhibiting the mimic, resulting in upregulation of the gene target.
In experiments in which a reporter vector will be used for downstream analysis, the vector, mimic, and inhibitor should all be cotransfected together and, in parallel, the vector and mimic should be cotransfected (see “miRNA mimic experiments — positive control”). Transfection of vector, mimic, and inhibitor should result in an increase in expression when compared to transfection of vector and mimic. This confirms that the inhibitor is effectively inhibiting the mimic, resulting in upregulation of the reporter.
miRNA inhibitor experiments — negative control
A negative control should be transfected in every inhibitor experiment and will indicate if results are nonspecific. Results achieved after transfection of this control should be similar to results from untransfected cells. Comparison of results from the negative control with results from the inhibitor under study can be used to confirm that the observed results are specific to the inhibitor under study.
In experiments in which a reporter vector will be used for downstream analysis, the negative control should be a cotransfection of the inhibitor negative control and the reporter vector.