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Designing Controls for siRNA ,,, Experiments

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 ch
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