Guidelines for transfection

Performing appropriate miRNA control experiments

The 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.
Transfection of a positive control mimic can be used to confirm that the experimental system is working as expected (i.e., that the mimic is efficiently transfected and causes downregulation of the target). This control can also be used in optimization experiments where varying concentrations are used for transfection to determine the concentration that provides optimal results. A positive control should be routinely transfected in every experiment using miRNA mimics to confirm that conditions remain optimal.
A negative control should be transfected in every experiment and will indicate if results are nonspecific. Comparison of results from the negative control with results from the miRNA mimic under study can be used to confirm that the observed results are specific to the miRNA mimic under study. Results from the negative control should also be compared to results from untransfected cells. Gene expression and phenotype should be similar in both untransfected cells and cells transfected with the negative control. Since miRNA mimics and siRNAs are chemically very similar and usually differ only in sequence, a negative control siRNA can also be used as a negative control miRNA mimic.

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.

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.

For optimal results, transfection efficiency should be as high as possible. When performing start-up experiments or working with a new cell line, it is necessary to perform multiple transfections under different conditions to determine the optimal conditions for maximum transfection efficiency. Transfection conditions that result in the greatest degree of cell death in comparison to transfection with a negative control can be maintained in future experiments. This control should be performed for optimization and start-up experiments and can be used as a routine transfection control in every experiment.
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