Performing appropriate RNAi control experiments

It is important to perform suitable control experiments so that results can be correctly interpreted. We recommend the following control experiments.

This is an siRNA that is known to provide high knockdown of its target gene. A positive control is used to establish that the experimental set up for transfection and knockdown analysis is working optimally. An siRNA that knocks down a gene resulting in the phenotypic effect under study may also be used as a positive control to ensure that the phenotypic assay is working optimally. A positive control siRNA should be transfected in every RNAi experiment.

A negative control siRNA should be a nonsilencing siRNA with no homology to any known mammalian gene. Transfection of negative control siRNA is used to determine whether changes in phenotype or gene expression are nonspecific. A negative control siRNA should be transfected in every RNAi experiment.

This control is used to measure the transfection efficiency. Transfection efficiency can be measured in several ways, for example by fluorescence microscopy after transfection of a fluorescently labeled siRNA or by observation of the level of cell death after transfection of siRNA that targets essential cell survival genes. siRNA transfection efficiency should be as high as possible. This control should be performed for optimization, for example, when establishing RNAi in a new cell line.

Mock-transfected cells go through the transfection process without addition of siRNA (i.e., cells are treated with transfection reagent only). This control is used to determine any nonspecific effects that may be caused by the transfection reagent or process.

Gene expression analysis should be carried out on cells that have not been treated to allow measurement of the normal, basal level of gene expression. Results from untreated cells can be used for comparison with results from all other samples. Untreated cells should be analyzed in every RNAi experiment.