Understanding siRNA and how to use it for RNAi
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.
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 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).
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.
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.
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.
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 . 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 . 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.
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 . 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 . All of these strategies, when combined, work efficiently to reduce off-targeting and to achieve potent and specific silencing for a successful RNAi experiment.
siRNAs are widely used to assess the individual contributions of genes to an assortment of cellular phenotypes including cytokinesis, apoptosis, insulin signaling [13, 14] and cell differentiation . siRNA screens have been used to identify novel pathways  and have had significant impact in validating targets for a number of cellular processes and diseases including cancer [17, 18], HIV infection  and hepatitis . Finally, in vivo RNAi 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 .
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.
Chemically synthesized siRNA reagents that target every gene in human, mouse and rat genome are available for convenient delivery in vitro.
For large scale siRNA screens, C911 controls can be a useful approach when carrying out in-depth hit confirmation.
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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Dharmacon siRNA products are the result of scientific innovation in siRNA design and novel modification strategies to optimize potency, specificity and delivery.
Our online Library Plater offers the flexibility to configure a library for your unique experimental needs.
Use our online design tools and extensive synthesis options to create a custom siRNA specific for your application.
Reagents optimized for your specific application.
Developing an understanding of a gene’s contribution to a particular phenotype is problematic when the protein has an extended half life.
This protocol outlines resuspension of siRNA and includes answers to frequently asked questions.
Bioinformatics, novel chemical modifications, and siRNA pooling significantly decrease off-target effects.
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