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Tips for Experimental Design

Carefully Choose Your siRNA Sequence for Maximum Specificity.
Although data indicate that siRNAs are highly specific, it is important to minimize the possibility of off-target effects by designing siRNAs that have limited sequence similarity to genes other than their intended target. Currently, it is not clearly understood how the number and location of mismatches between the antisense siRNA strand and the target mRNA affect silencing. Some reports indicate that a single mismatch in the center of an siRNA sequence can abolish silencing effects (1,2). In fact, this remarkable specificity has been shown in a model system to permit allele specific gene silencing (2). In contrast, another report indicates that siRNAs with regions of 14-15 contiguous bases of sequence similarity between the siRNA and an mRNA can induce silencing (3). Until we develop a better understanding of the specificity of siRNAs for their targets, it is best to design siRNAs that are completely complementary to the mRNA target and that contain at least 2 or more mismatches to all off target mRNAs. Also, results obtained with pools or populations of siRNAs should be confirmed with individual siRNAs to enhance the confidence level in the data.

Use the Minimum siRNA Concentration Required to Achieve Silencing.
Recent reports indicate that siRNA concentrations of 100 nM or higher in mammalian cultured cells can lead to nonspecific changes in gene expression (3,4). In general, transfecting 5-20 nM of an efficacious siRNA appears to minimize nonspecific effects while providing maximal target gene silencing. The concentration of siRNA required will depend upon the method and efficiency of siRNA delivery, the cell line used, and the effectiveness of the siRNA sequence.

Suggested Experimental Controls

Ambion scientists use and recommend a number of different controls for siRNA experiments. Most of these coincide with the suggested controls detailed in a recent editorial published in Nature Cell Biology (5). These controls can be used in virtually any gene silencing experiment, whether the effects are analyzed one gene at a time using more traditional mRNA and protein analysis techniques or on a genome-wide scale using microarrays.

Scrambled siRNA Control
A scrambled siRNA control is used to discount any changes to the gene expression profile that may result from the siRNA delivery method. Comparing cells transfected with a scrambled siRNA control to untransfected cells can reveal changes caused by siRNA delivery.

Positive siRNA Control
Ambion scientists always include a positive siRNA control in their experiments -- usually the Silencer GAPDH siRNA -- which serves to monitor siRNA transfection efficiency. When the positive control fails to elicit the expected reduction in gene expression, poor transfection is immediately suspected.

Multiple siRNAs to a Single Target
One of the best ways to increase confidence in data from siRNA experiments is to use two or more siRNAs to a single gene target. The siRNAs should first be analyzed for effectiveness at reducing target gene expression. Different siRNAs to the same gene with comparable gene silencing efficacy should induce similar changes in gene expression profile and phenotype. Any changes induced by one siRNA and not the other(s) could be attributed to off target effects. This approach is also valid for siRNA cocktails. In other words, cocktails prepared from different regions of the same gene can be used to confirm gene silencing results.

Monitor the Antiviral Response
Recent evidence indicates that up-regulation of the antiviral response may be a useful indicator of nonspecific siRNA effects. Although we do not yet routinely monitor the antiviral response in our own experiments, there are several published reports on how this response can be detected. One way is to assess levels of 2'5'-oligoadenylate synthetase mRNA (6). 2'5' oligoadenylate synthetase catalyzes the synthesis of 2'-5' polyadenylic acid, which activates the nonspecific ribonuclease RNase L. Another way is to monitor activation of the dsRNA-dependent protein kinase, PKR, which is involved in the type 1 interferon response. PKR phosphorylates the small subunit of the eukaryotic initiation factor 2-alpha (eIF2alpha), which results in nonspecific inhibition of translation. PKR activation can therefore be examined by monitoring the phosphorylation of eIF2alpha (7).

Monitor Both Target mRNA and Protein Levels
In siRNA experiments it may be beneficial to monitor both mRNA and protein levels for several reasons. For instance, mRNA reduction seen without a corresponding reduction in protein levels can indicate that protein turnover is slow. Furthermore, protein reduction in the absence of mRNA reduction may indicate that an siRNA is mediating its effects at the translational level like a microRNA.

The use of RNAi has enormous potential for analyzing gene function, elucidating biological pathways, and identifying and validating potential drug targets. The RNAi field is still in its infancy, and recommendations on experimental design and proper controls are likely to evolve. Ambion is committed to bringing researchers around the world the most up-to-date information on RNAi for their own research. For the latest information, see the RNAi Resource.

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