siRNA Applications

Understanding siRNA and how to use it for RNAi. Small (or short) interfering RNA (siRNA) is the most commonly used RNA interference (RNAi) tool for inducing short-term silencing of protein coding genes.

siRNA Products

What is siRNA?

siRNA is a synthetic RNA duplex designed to specifically target a particular mRNA for degradation. While siRNA provides the opportunity to induce gene knockdown in a variety of cell lines, their utility is limited to cells that are amenable to transfection of synthetic oligonucleotides.

Since siRNAs achieve transient silencing, experiments are limited to relatively short time frames on the order of 2-4 days. siRNAs can also be used for knockdown of non-protein coding genes, such as long noncoding RNAs (lncRNA).

Video animation: RNA interference

RNA interference (RNAi) is an important pathway that is used in many different organisms to regulate gene expression. This animation introduces the principles of RNAi involving small interfering RNAs (siRNAs) and microRNAs (miRNAs). We take you on an audio-visual journey through the steps of gene expression and show you an up-to-date view of how RNAi can silence specific mRNAs in the cytoplasm.

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Structure of siRNA

applications sirna general structure of sirna

Figure 1. General structure of siRNA. Two RNA strands form a duplex 21 bp long with 3' dinucleotide overhangs on each strand. The antisense strand is a perfect reverse complement of the intended target mRNA.

How does siRNA work?

siRNAs consist of two RNA strands, an antisense (or guide) strand and a sense (or passenger) strand, which form a duplex 19 to 25 bp in length with 3' dinucleotide overhangs (Figure 1).

Synthetic siRNAs are most commonly generated through solid-phase chemical synthesis methods (such as patented 2'-ACE chemistry) which provide highly pure, stable, and readily modified siRNAs. Small double-strand siRNAs are transfected into cells where the guide strand is loaded into RISC. This activated protein and nucleic acid complex can then elicit gene silencing by binding, through perfect complementarity, to a single target mRNA sequence, thereby targeting it for cleavage and degradation.

How is siRNA delivered to a cell?

siRNA must be transfected into cells either by cationic lipid or polymer-based transfection reagents, electroporation (physical delivery via plasma membrane holes created by an electrical field), or adding chemical modifications to the duplex to aid in uptake by the cell.

Table 1 lists the advantages and limitations of each delivery method.

Delivery Mode

Advantages

Disadvantages

Transfection

  • Cationic liposomes or polymer based
  • Delivery of siRNA, microRNAs, and shRNA into most cell types
  • Not all cell types amenable to transfection reagents

Electroporation

  • Electrical pulse
  • Effective for difficult-to-transfect cells
  • Cell death often increased

Viral-mediated Delivery

  • Lentivirus
  • Retrovirus
  • Adeno-associated virus
  • Effective for difficult-to-transfect cells
  • For use in stable selection
  • In vivo application
  • Requires BSL2 facilities
  • May trigger antiviral response in some cell types

Modified siRNA

  • Modified siRNA (Accell)
  • To enable passive uptake by many cell types
  • Effective for difficult-to-transfect cells
  • Repeated dosing possible for longer-term silencing
  • In vivo application
  • Delivery efficiency inhibited by presence of >3% serum during application

Table 1. The primary considerations in selecting a delivery method for siRNA are the suitability of the method to the cells and the assay requirements for duration of silencing.Technique

siRNA Function

The success of RNAi experiments depends on the efficiency of gene knockdown. Early work on siRNA design established conventional guidelines for siRNA structural attributes that led to reasonable functional knockdown in specific cases [1].

The properties of potent siRNAs were further refined by performing large-scale functional studies that defined thermodynamic and sequence-based rules for rational siRNA design [2]. These design algorithms greatly improved the reliability of identifying potent siRNA sequences. The Dharmacon SMARTselection algorithm was the first comprehensive rational design strategy applied to commercial collections. While research is continually striving to identify molecules with greater activity and specificity, siRNAs designed by SMARTselection strategies, such as siGENOME and ON-TARGETplus reagents, remain the most effective reagents on the market.

Specificity

Although the sequence complementarity-based mechanism underlying RNAi allows for target-specific gene knockdown, the same mechanism can result in unintended knockdown of genes not being directly targeted. Several strategies have been developed to mitigate these so-called "off-target" effects and ensure on-target activity.

Chemical modifications to the siRNA have been used successfully to promote preferential loading of the intended antisense (guide) strand into the RISC complex [3, 4] and reduce sense (passenger) strand loading and activity [5, 6]. Further, to reduce the risk of the siRNA guide strand seed region from causing off-target effects, design algorithms can incorporate filters that exclude high-frequency seed sequences from known mammalian microRNAs [7]. Chemical modifications or thermodynamic-based design considerations can also be applied to the siRNA seed region to discourage undesired interactions [5, 8, 9]. Finally, the strategy of pooling several independent siRNAs that target an individual gene has been shown to reduce the total number of non-specific gene targets and the frequency of off-target phenotypes while preserving potent target gene knockdown [10]. All of these strategies, when combined, work efficiently to reduce off-targeting and to achieve potent and specific silencing for a successful RNAi experiment.

Applications

siRNAs are widely used to assess the individual contributions of genes to an assortment of cellular phenotypes including cytokinesis[11], apoptosis[12], insulin signaling [13, 14] and cell differentiation [15].

siRNA screens have been used to identify novel pathways [16] and have had significant impact in validating targets for a number of cellular processes and diseases including cancer [17, 18], HIV infection [19] and hepatitis [20]. Finally, in vivoRNAi has been used for target validation studies in animal disease models and has the potential to be used for therapeutic purposes where disease-causing genes are selectively targeted and suppressed [21].

Controls for siRNA Experiments

Controls are an essential part of every siRNA experiment. At least three types of controls should be used in each siRNA (and RNAi) experiment: positive control, negative control and untreated control.

A well-characterized positive control allows the researcher to ensure the delivery method is sufficient to achieve effective silencing. Negative controls help to separate sequence-specific effects from the effects of experimental conditions on cellular responses. An untreated control establishes a useful baseline reference for cell phenotypes and gene expression levels.

Effective Controls for RNAi - Tech Note

  • Chemically synthesized siRNA reagents that target every gene in human, mouse and rat genome are available for convenient delivery in vitro.

C911 Controls for siRNA Screening

  • For large scale siRNA screens, C911 controls can be a useful approach when carrying out in-depth hit confirmation.

Dharmacon siRNA Selection Guide

figure- mage sirna selection guide

We offer a wide selection of predesigned siRNA product lines and formats. This quick selection guide will help to determine the best option for your particular needs.

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Featured Products

siRNA - Products

Dharmacon siRNA products are the result of scientific innovation in siRNA design and novel modification strategies to optimize potency, specificity and delivery.

Cherry-pick Libraries

  • Our online Library Plater offers the flexibility to configure a library for your unique experimental needs.

Custom siRNA Synthesis

  • Use our online design tools and extensive synthesis options to create a custom siRNA specific for your application.

Transfection - Products

  • Reagents optimized for your specific application.

Featured Resources

Accell siRNA Reagents: Achieving Long-term Gene Silencing - Application Note

  • Developing an understanding of a gene’s contribution to a particular phenotype is problematic when the protein has an extended half life.

Basic siRNA Resuspension - Protocol

  • This protocol outlines resuspension of siRNA and includes answers to frequently asked questions.

Off-Target Effects: Disturbing the Silence of RNA interference - Technical Note

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

References

  1. Elbashir, S.M., et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001. 411: p. 494-8.
  2. Reynolds, A., et al., Rational siRNA design for RNA interference. Nature Biotechnology, 2004. 22(3): p. 326-330.
  3. Khvorova, A., A. Reynolds, and S. Jayasena, Functional siRNAs and miRNAs exhibit strand bias. Cell, 2003. 115: p. 209-216.
  4. Schwarz, D.S., et al., Asymmetry in the assembly of the RNAi enzyme complex. Cell, 2003. 115(2): p. 199-208.
  5. Jackson, A.L., et al., Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing. Rna, 2006.
  6. Vaish, N., et al., Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs. Nucleic Acids Res., 2011. 39(5): p. 1823-1832.
  7. Birmingham, A., et al., A protocol for designing siRNAs with high functionality and specificity. Nat Protoc, 2007. 2: p. 2068-78.
  8. Bramsen, J.B., et al., A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects. Nucleic Acids Res., 2010. 38(17): p. 5761-5773.
  9. Ui-Tei, K., et al., Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect. Nucleic Acids Res., 2008. 36(22): p. 7100-7109.
  10. Kittler, R., et al., Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nat Methods, 2007. 4: p. 337-44.
  11. Zhou, T., et al., A role for Plk1 phosphorylation of NudC in cytokinesis. Dev Cell, 2003. 5(1): p. 127-38.
  12. Zender, L., et al., Caspase 8 small interfering RNA prevents acute liver failure in mice. PNAS, 2003. 100(13): p. 7797-7802.
  13. Hsieh, A.C., et al., A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens. Nucleic Acids Res, 2004. 32: p. 893-901.
  14. Jiang, Z.Y., et al., Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. PNAS, 2003. 100: p. 7569-7574.
  15. Kurisaki, K., et al., Nuclear Factor YY1 Inhibits Transforming Growth Factor {beta}- and Bone Morphogenetic Protein-Induced Cell Differentiation. Mol. Cell. Biol., 2003. 23: p. 4494-4510.
  16. Struwe, W.B. and C.E. Warren, High-throughput RNAi screening for N-glycosylation dependent loci in Caenorhabditis elegans. Methods Enzymol, 2010. 480: p. 477-93.
  17. Whitehurst, A.W., et al., Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature, 2007. 446(7137): p. 815-9.
  18. Bric, A., et al., Functional identification of tumor-suppressor genes through an in vivo RNA interference screen in a mouse lymphoma model. Cancer Cell, 2009. 16(4): p. 324-35.
  19. Kok, K.H., T. Lei, and D.Y. Jin, siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication. Retrovirology, 2009. 6: p. 78.
  20. Li, Q., et al., A genome-wide genetic screen for host factors required for hepatitis C virus propagation. PNAS, 2009. 106: p. 16410-16415.
  21. Dominska, M. and D.M. Dykxhoorn, Breaking down the barriers: siRNA delivery and endosome escape. J Cell Sci, 2010. 123(Pt 8): p. 1183-9.