RNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism mediated by non-coding RNA molecules known as microRNAs. miRNAs can target hundreds of genes simultaneously (1), inducing subtle but reproducible shifts in gene expression and regulation.
This mechanism can be utilized for targeted gene silencing by introduction of nucleic acid based tools that are specially designed to trigger the RNAi mechanism. These molecules are taken up by the RNA induced silencing complex (RISC) to target specific microRNAs for degradation or to modulate endogenous microRNAs.
Currently, there are three primary categories of RNAi tools to perform these functions: small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA mimics and inhibitors.
- Benefit: Proven and cost-effective, siRNA is the most commonly used RNAi tool for inducing transient gene silencing
- How it works: A synthetic, double-strand RNA duplex of 19-21 nt in length is transfected into cells and targets a specific miRNA for degradation
- Learn more: About the basics of how siRNA works, and how to use it experimentally on our siRNA applications page.
- Benefit: shRNA is most useful for longer-term gene silencing and delivery into difficult-to-transfect cell types
- How it works: A shRNA vector is delivered via plasmid transfection or viral transduction for endogenous expression. The expressed stem-loop structures are then processed by the cell into siRNAs.
- Learn more: About the basics of how shRNA works, and how to use it experimentally on our shRNA applications page.
- Benefit: Synthetic or expressed microRNAs are valuable for studying microRNAs by gain- or loss-of-function assays
- How it works: Synthetic microRNA inhibitors are designed to bind native microRNAs to prevent their activity, thereby providing loss-of-function results. Expressed microRNA mimics may be placed under an inducible promoter for temporal studies of microRNA roles
- Learn more: About the basics of how microRNA works, and how to use it experimentally on our microRNA applications page.
These tools have enabled a shift from traditional gene knockout, antisense or ribozyme technologies towards RNAi as a standard technique for discovery biology and target validation. RNAi is routinely manipulated to perform individual gene functional analyses, as well as to dissect complex biological pathways or survey entire genomes in a high-throughput manner.
Harnessing the endogenous microRNA pathway
A schematic of the endogenous microRNA pathway, with points of entry for the three categories of RNAi tools.The endogenous microRNA pathway begins with genomic DNA transcription to produce pri-microRNA, which is then processed by the microprocessor complex containing the DROSHA protein and other proteins to a pre-microRNA hairpin structure and exported from the nucleus by Exportin-5. In the cytoplasm, the pre-microRNA is further processed by the Dicer complex to double-strand anti-parallel RNA. One strand of this RNA duplex will load into the RISC complex and act to target a mRNA transcript for down-regulation by either (1) seed-mediated mRNA translational repression or (2) catalytic mRNA cleavage mediated by high sequence complementarity. The blue molecules to the right of the image illustrate different classes of RNAi research tools and where they enter the endogenous RNAi pathway.
- Dharmacon RNAi products encompass the most complete portfolio of innovative tools for transient, long-term, inducible and in vivo RNAi applications.
- Enjoy optimal specificity from the only siRNA with a patented modification pattern to reduce off-targets caused by either the sense or the antisense strand.
- Fast and easy online configuration and ordering of plated siRNA and microRNA reagents targeting your genes of interest.
- The most advanced and flexible single-vector inducible shRNA available for tightly controlled gene silencing
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- The information provided in this document represents anecdotal customer feedback.
- Chemically synthesized siRNA reagents that target every gene in human, mouse and rat genome are available for convenient delivery in vitro.
- Bioinformatics, novel chemical modifications, and siRNA pooling significantly decrease off-target effects.
- Friedman, R.C. et al. Most mammalian mRNAs are conserved targets of microRNAs. Genome Research 19(1):92-105 (2009).