Controls for RNAi Experiments

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).

• View and order Silencer Select siRNA negative controls
• View and order Stealth RNAi negative controls
• View and order Silencer siRNA negative controls

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).

• View and order Silencer Select siRNA positive controls
• View and order Stealth RNAi positive controls
• View and order Silencer siRNA positive controls

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.

• View and order fluorescently labeled transfection controls
• Learn more about RNAi transfection

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.

Silencer Select negative controls

• Non-targeting controls with no significant sequence similarity to mouse, rat, or human gene sequences
• Chemically modified for reduced off-target effects as found in other Silencer Select siRNAs
• Tested by microarray analysis and shown to have minimal effects on gene expression
• Assessed in multiparametric cell-based assays and are proven to have no significant effect on cell proliferation, viability, or morphology in the cell lines tested.

Silencer Select siRNA positive controls

• Extensively validated for optimizing and monitoring transfection efficiency in human, mouse, and rat cells.
• Functionally tested in several common cell lines
• Include Silencer Select siRNA modifications for enhanced specificity

Stealth RNAi siRNA negative controls

• Negative controls that offer low, medium, and high levels of GC content to match that of the experimental Stealth RNAi siRNAs
• Medium GC is recommended for experiments using numerous unique siRNA designs

Stealth RNAi siRNA positive controls

• Validated reagents targeting housekeeping genes or common reporter genes
• Ideal as a control for assessing knockdown and optimizing RNAi experiments

Fluorescently labeled transfection controls

An siRNA or oligo with a fluorophore conjugate, such as FAM or Cy3, can be useful as a visual indicator of transfection success, especially during transfection optimization when conditions such as cell density and the amount of transfection reagent are being varied.It should be noted that intracellular fluorescence is not a substitute for quantitation of transfection efficiency as measured by knockdown from a validated positive control.