High-throughput screening with RNAi libraries has provided researchers with an unbiased approach for identifying novel genes involved in biological processes. This method has been used successfully for synthetic lethality screens in cancer cells, identification of host factors exploited by pathogens, and a variety of basic biological screens (for examples, see our list of recommended RNAi Screening References).
Due in part to the potential for off-target effects, one of the most time-consuming steps in the screening workflow is validation of hits identified in the initial screen. A combination of experimental and bioinformatic approaches are typically used to narrow down the list of hits to a manageable number of targets that can be studied in depth. Screeners generally agree that collecting data from multiple independent siRNA sequences, multiple assays, multiple cell lines, and/or complementary techniques are important for generating a list of high-confidence hits. A greater number of data points can help to simplify the stratification of hits.
One experimental tool for identifying off-targets (false positives) in a siRNA screen is a seed-matched control. These controls are designed to contain the same seed sequence (nt 2-8 of the antisense strand) as the original siRNA in the screen with all or a portion of the remaining sequence altered to prevent target gene knockdown. C911 controls  are a version of seed-matched controls that are quickly growing in popularity.
A C911 control has the same sequence as the experimental siRNA used in the original screen except the bases in positions 9, 10 and 11 are changed to their complement. The mismatches are thought to disrupt cleavage of the intended mRNA target, but still maintain potential off-target effects. Comparison of the original siRNA and the C911 control in a phenotypic assay should distinguish results that are due to down-regulation of the intended target rather than off-target effects.
A C911 control has bases in positions 9, 10 and 11 changed to their direct complement compared to the targeting siRNA sequence.
The C911 design is simple to generate manually for a small number of siRNAs. For screeners working with a larger list of siRNAs, the authors of the C911 publication have developed a tool designed to quickly convert your original siRNA target sequences into C911 control target sequences. To design C911 controls for Dharmacon siRNAs using this tool, please follow these steps:
Example of C911 control sequence generation using the C911 Calculator available from the NIH. The sequence in the column labeled "C911 Target" is the input for custom synthesis to generate a C911 control based on the input experimental siRNA sequence.
C911 controls must be ordered as custom siRNAs. These orders can be placed using our online custom siRNA ordering tool.
Alternatively, a list of C911 sequences may be provided directly to Customer Support. Please provide desired siRNA names, processing, overhangs, and synthesis scale.
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Dharmacon RNAi products encompass the most complete portfolio of innovative tools for transient, long-term, inducible and in vivo RNAi applications.
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