Negative control siRNAs are most often a non-targeting siRNA - designed not to target any gene - for determining the non-specific effects of siRNA delivery and for providing a baseline to compare to siRNA-treated samples. Negative controls may also be a functional siRNA that is known to have no impact on your phenotype of interest, which should be determined empirically.
We recommend using one or more negative controls in every RNAi experiment. You should match the control siRNA to the experimental siRNA type (for example, use only Silencer Select siRNA controls for experiments using Silencer Select siRNA).
Positive controls provide confidence in your RNAi experiments by confirming that experimental conditions were met to achieve robust silencing. Positive controls are validated siRNAs that are known to achieve high levels of knockdown (>70%), typically for a constitutively expressed or housekeeping gene. A positive control should be used to optimize siRNA delivery conditions and to reconfirm high levels of delivery in each RNAi experiment. When a positive control fails to produce the anticipated phenotype, carefully evaluate your experimental conditions and decide if some factors need to be adjusted.
You should match the control siRNA to the experimental siRNA type (for example, use only Silencer Select siRNA controls for experiments using Silencer Select siRNA).
To achieve the highest transfection efficiency possible, it is advised to first optimize transfection conditions for your cell lines. Fluorescently labeled RNAs can help with this by providing a qualitative, visual indicator of transfection success. Keep in mind that it is also important to monitor transfection in each experiment with a validated positive control siRNA for knockdown.
For optimal siRNA transfection, we have many cell type-specific transfection protocols for siRNA delivery using Lipofectamine RNAiMAX Transfection Reagent to help you get started.
In addition to the proper use of controls, these guidelines outline experimental best-practices for RNAi gene silencing.
Correlate phenotypes with reduced target mRNA levels
A phenotypic assay is often used as an indicator of a successful RNAi-mediated loss-of-function experiment. However, an observed change in your cells due to target gene silencing should be correlated with the loss of the corresponding mRNA levels using qRT-PCR. If one or more of the gene-targeting siRNAs gives a phenotype but not a loss in mRNA, then the phenotype could be due to a non-specific response or off-target gene silencing.
Use multiple siRNA sequences to the same target
siRNA sequences with partial homology to other targets may contribute to off-target activity. Gene profiling experiments have shown that duplexes with partial homology to other transcripts can cleave the target or act like a microRNA (miRNA), inhibiting translation of the target mRNA. To ensure that knockdown of the intended gene can be attributed to the observed phenotype, the results should be confirmed by at least two unique siRNA reagents that target non-overlapping regions of the target mRNA.
Titration of siRNA
Silencer Select siRNA and Stealth RNAi siRNA can be very effective even at low concentrations, so you should aim to use the lowest effective level to avoid altering the cells’ normal processes. Titrating down the dose of the siRNA enables you to reduce off-target or nonspecific effects while achieving robust knockdown.
Untransfected controls
Cells that are not transfected or treated with siRNA are an important control for the overall reproducibility of your experiment. These cell populations control for the impact of media changes,reactivity to assay conditions, or other variables, while providing a secondary baseline for negative/non-targeting controls in determination of cell viability.
Downstream detection assays
Before transfecting cells with siRNA, we recommend validating the reagents that will be used in qRT-PCR and western blots to measure mRNA and protein levels. Validating qRT-PCR primers or antibodies for your positive control and target genes before performing knockdown experiments ensures that these reagents are sensitive enough to detect changes in your target gene’s expression due to knockdown. Without sufficient sensitivity, it can be difficult to interpret knockdown results from genes or proteins with low expression levels.