siRNA: To pool, or not to pool?

What factors should be considered in making the decision between using an siRNA individual or pool?

For maximum experimental flexibility, Dharmacon offers both individual siRNA (a single duplex) and a pooled siRNA reagent (mixture of four different siRNAs targeting a single gene). What does this mean, and what factors should be considered in making a decision between an siRNA individual and a pool?

The Dharmacon strategy is to design four individual siRNAs for every human, mouse and rat gene, using a highly validated, rational design algorithm. A SMARTpool™ siRNA reagent is a mixture of these 4 siRNAs provided in a single tube. When making the decision between using multiple individual siRNAs or a single SMARTpool reagent, consider these few points to help determine which format may be best suited to your experimental objectives:

Whole gene vs. transcript-specific knockdown

SMARTpool reagents are designed to target all transcript/splice variants associated with a single gene as recorded in the RefSeq database. Therefore, isoforms arising from splice variants would all be targeted by the SMARTpool for that gene to ensure complete knockdown. Additionally, the 4 sequences in the SMARTpool will target multiple regions within the transcript, including both the UTRs and ORF, for very complete knockdown. If single variants or specific regions need to be targeted (as when designing rescue experiments or for functional studies of particular isoforms), using individual siRNA sequences would be advised due to the very specific design requirements.

Large-scale RNAi screen

A key benefit of using a SMARTpool siRNA reagent is a reduction of off-target effects. Additionally, the knockdown efficiency of a SMARTpool typically mirrors that of the most functional siRNA design in the pool. Together, these benefits allow researchers to use a single reagent per gene. This is especially advantageous when using siRNAs for screening purposes, as they result in fewer false positives and false negatives (respectively) while reducing the amount of assay reagents and consumables needed when compared to screening with multiple individual siRNAs per gene. Additional research also suggests that pooled screening conditions can also favor increased phenotypic penetrance and the ability to see loss-of-function phenotypes, as detailed in Parsons et al.1 In addition, the four component siRNAs of a SMARTpool can be used individually as independent confirmation of a phenotype following the initial screen, fulfilling the best-practice of hit confirmation with multiple independent siRNAs.

In vitro vs. in vivo assays

Dharmacon pre-designed SMARTpool siRNA reagents combine the power of four predesigned duplexes in a single tube, allowing researchers working in vitro to achieve guaranteed silencing using just one reagent. However, in vivowork typically requires quantities and purification levels achievable only with custom synthesis. For this reason, many researchers find it most economically feasible to do an in vitro pilot study with multiple individual siRNAs to determine the best single sequence, then carry forward with the more precious animal model work using a single, verified individual siRNA. For more information on important considerations for in vivo experimentation see our Technical Note: "In vivo RNAi: Biodistribution, Delivery, and Applications"

References

  1. B.D. Parsons, A. Schindler, D.H. Evans, E. Foley, A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay. PLoS One. 4(12), e8471 (2009).

Additional Resources

Dharmacon siRNA Selection Guide

  • A quick tool to help determine the ideal Dharmacon siRNA product line and format for your needs.

In vivo RNAi: Biodistribution, Delivery, and Applications - Tech Note

  • The dsRNA is cleaved by the RNase-III enzyme, Dicer, into small interfering RNAs (siRNA) that are approximately 21-23 nt.

Off-target Effects: Disturbing the Silence of RNA interference (RNAi) - Tech Note

  • Bioinformatics, novel chemical modifications, and siRNA pooling significantly decrease off-target effects.