The Dharmacon SMARTselection algorithm was the first comprehensive rational design strategy applied to commercial collections. 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. 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 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. 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. View PDF ». Dharmacon siRNA products are the result of scientific innovation in siRNA design and novel modification strategies to optimize potency, specificity and delivery. Contact Us. View all applications. How can we help you? Need help? What is siRNA? How does siRNA work? How is siRNA delivered to a cell?
Table 1 lists the advantages and limitations of each delivery method. 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. 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].
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. Custom siRNA synthesis Use our online design tools and extensive synthesis options to create a custom modified siRNA specific for your application. Bartel, D. MicroRNAs: target recognition and regulatory functions. Cell , — Baulcombe, D. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants.
Plant Mol. Behm-Ansmant, I. Genes Dev. Bernstein, E. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature , — Birmingham, A. Methods 3, — Bohnsack, M. Rna 10, — Bologna, N. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev. Plant Biol. Cell 69, —e5. Google Scholar. Braun, J. Brodersen, P. Science , — Buehler, E. Sci Rep. Cai, Q. Cai, X. Carbonell, A. Methods Mol. Das, S. Denli, A. Processing of primary microRNAs by the Microprocessor complex.
Doench, J. Dorsett, Y. Drug Discov. Dueck, A. Assembly and function of small RNA - Argonaute protein complexes. Elbashir, S. Elmen, J. Nucleic Acids Res. Fedorov, Y. Off-target effects by siRNA can induce toxic phenotype. Rna 12, — Filipowicz, W. Cell , 17— Fire, A. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Frank, F. Embo J. Fukudome, A. Plant Res. Gregory, R. The Microprocessor complex mediates the genesis of microRNAs.
Grishok, A. Cell , 23— Han, J. Hannus, M. Iki, T. Ipsaro, J. From guide to target: molecular insights into eukaryotic RNA-interference machinery. Iwasaki, S. Cell 39, — Iwasaki, Y. Jackson, A. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Jakymiw, A. Cell Biol. Jonas, S. Towards a molecular understanding of microRNA-mediated gene silencing. Ketting, R. Kim, V. Biogenesis of small RNAs in animals. Kittler, R. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells.
When a mammal cell is faced with a double-stranded RNA such as a siRNA, it may mistake it as a viral by-product and initiate an immune response. In addition, the introduction of a siRNA may cause unintended off-targeting where other non-threatening proteins may also be attacked and knocked out.
Introducing too much siRNA to the body can result in nonspecific events due to innate immune response activation, but given the ability to beat any gene of interest, siRNAs have the potential for many therapeutic uses. Many diseases can potentially be treated by inhibiting gene expression, by chemically modifying siRNAs to enhance their therapeutic properties.
Some properties that could be enhanced are:. Therefore, the design of synthetic siRNA for therapeutic uses has become a popular objective of many biopharmaceutical companies. A detailed database of all such chemical modification is manually curated at siRNAmod , a manually curated database of experimentally validated chemically modified siRNAs.
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