Identification of Appropriate Experimental Controls
To ensure that the conclusions drawn from siRNA experiments are accurate, it is necessary to include the appropriate experimental controls. We recommend including at least four types of experimental controls in every RNAi experiment. Parallel testing of multiple controls under several conditions can be easily performed using the 96-well Shuttle System.
1) Positive siRNA control: This should be a validated siRNA pool or individual siRNA targeting a well-characterized housekeeping gene, such as cyclophilin B (also known as PPIB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or Lamin. A good positive control reagent targeting a well-expressed but non-essential gene is useful for establishing experimental parameters without affecting cellular viability and can also be used as negative control that is unassociated with any particular pathway under study (i.e., it fails to generate a observable phenotype in the assay being employed).
2) Negative siRNA control: Negative siRNA control reagents are bioinformatically designed to have no known target in the cell line of choice. These reagents are important for distinguishing sequence-specific silencing from sequence-independent effects that are associated with the delivery of siRNA into the cell. Such sequence independent effects can include toxicity resulting from the process of transfection in conjunction with nucleic acid delivery or hyper-sensitivity to introduction of double stranded RNA. Investigators are encouraged to test multiple candidates in their own experimental systems to empirically confirm that the negative controls do not result in any observable and unintended off-target effects. For that purpose 
Dharmacon offers a comprehensive portfolio of multiple negative controls, including the ON-TARGETplus™ Non-Targeting Controls, which have been confirmed by microarray analysis to have little to no off-target signature in HeLa cells.
3) Untreated transfection control: The untreated control sample is comprised of cells that have neither been treated with siRNA nor subjected to the transfection process. This control serves as an indicator of baseline cellular activity to which all other conditions can be compared.
4) Mock-treated control: The mock-treated control sample is one in which the cells are subjected to the transfection procedure in the absence of siRNA. In the case of nucleofection, the cells would be exposed to the Nucleofector Solution and subjected to the nucleofection procedure in the absence of siRNA. The analysis of mock-treated cells will indicate whether the transfection process results in cytotoxicity or other non-specific effects.
