What is shRNA and how do you use it?
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.
Short hairpin RNA (shRNA) sequences are usually encoded in a DNA vector that can be introduced into cells via plasmid transfection or viral transduction. shRNA molecules can be divided into two main categories based on their designs: simple stem-loop and microRNA-adapted shRNA. A simple stem-loop shRNA is often transcribed under the control of an RNA Polymerase III (Pol III) promoter [1, 2]. The 50-70 nucleotide transcript forms a stem-loop structure consisting of a 19 to 29 bp region of double-strand RNA (the stem) bridged by a region of predominantly single-strand RNA (the loop) and a dinucleotide 3' overhang [3-5]. The simple stem-loop shRNA is transcribed in the nucleus and enters the RNAi pathway similar to a pre-microRNA. The longer (> 250 nucleotide) microRNA-adapted shRNA is a design that more closely resembles native pri-microRNA molecules, and consists of a shRNA stem structure which may include microRNA-like mismatches, bridged by a loop and flanked by 5' and 3' endogenous microRNA sequences . The microRNA-adapted shRNA, like the simple stem-loop hairpin, is also transcribed in the nucleus but is thought to enter the RNAi pathway earlier similar to an endogenous pri-microRNA.
shRNA technologies are DNA-based, which provides flexibility in design of the vector. Most vector-based shRNA systems contain a selectable marker to allow for the elimination of cells that have not been successfully transfected or transduced, and maintenance of cells with sustained gene knockdown. The shRNA expression cassettes can also be incorporated into viral vector systems, including retrovirus, adeno-associated virus, adenovirus and lentivirus, which permit stable integration into and expression from the host genome. These viral strategies permit shRNA delivery to cell lines that are refractory to transfection. Fluorescent markers (such as a Green or Red Fluorescent Protein [GFP or RFP]) can also be included for tracking cells expressing shRNAs. The performance of shRNA is influenced by many factors including the efficiency of transduction or transfection, the promoter driving expression of the shRNA and epigenetic modifications (which can lead to silencing of shRNA expression). Further, the influence that each of these factors have on vector performance can differ depending on the cell line or cell type [7-9]. SMARTchoice promoter and reporter options are available to help researchers select optimal promoters for shRNA expression and gene silencing. Finally, when these shRNA expression cassettes are coupled with inducible promoters, as with SMARTvector Inducible Lentiviral shRNA or the TRIPZ vector system, researchers can design studies to temporally and spatially modulate gene expression [10-12].
There are two options for DNA-based shRNA delivery to cells. For plasmids, typical transfection methods such as use of lipid transfection reagents or electroporation can be employed. Lentiviral particles are an excellent choice for difficult to transfect cells, and in situations where high efficiency is needed or multiple constructs per cell need to be delivered. Most modern vectors have selectable markers that allow the selective killing of cells in the culture that were not successfully transfected or transduced, so that a pure culture can be developed.
Optimal gene knockdown is a requirement for successful RNAi using shRNA systems. Rational design of shRNA sequences has largely been based on algorithms developed with siRNA. While some siRNA design rules apply to shRNA, more refined shRNA design algorithms will likely improve target gene silencing for shRNAs in the future . In order to predict functional shRNA sequences, Dharmacon's SMARTvector™ shRNA algorithm selects target sequences on the basis of numerous criteria, including position-dependent nucleotide preferences, secondary structure and thermodynamic stability profiles specific to the SMARTvector microRNA-based scaffold. Additionally, the SMARTvector algorithm includes several criteria for increasing specificity.
As with siRNAs, bioinformatic approaches can be applied to create target-specific shRNAs while minimizing the potential for off-target effects. High concentrations of silencing intermediates are known to contribute to off-target events, but the level of the intermediates are difficult to control when shRNAs are exogenously expressed. Several publications have documented evidence that, under specific conditions, high levels of simple stem-loop shRNA expression may saturate the endogenous RNAi pathway, and lead to unintended phenotypes . Other studies have suggested that the use of a microRNA-adapted shRNA may reduce cellular toxicity for in vivo RNAi experiments [6, 15] due to these scaffolds being more efficiently processed by both Drosha-DGCR8 and Dicer [16, 17].
shRNA offers the possibility of prolonged gene silencing. Transduction of viral-based shRNA allows access to cells, such as primary and neuronal cells, that are difficult to transfect by traditional cationic lipid-based strategies. Viral-based shRNAs have also been used to evaluate gene function at whole genome scales using pools of silencing constructs. Pooled or arrayed screens are now widely used to perform high-throughput screens to identify genes required in a variety of processes such as cancer cell survival and proliferation [18-20], tumor suppressor pathway components , modulators of the mammalian circadian clock , suppressors of epithelial mesenchymal transition , host mediators of HIV-1 replication [24, 25], and regulators of cell migration . The power of pooled RNAi screening has also been extended to screening for gene function in animal models to investigate in vivo biology [27, 28].
We offer a selection of shRNA reagents and libraries. This quick selection guide will help to determine the best option for your particular needs.
Lentiviral vector-based reagents for RNA interference.
Make smart, informed choices for successful gene silencing in your cells of interest.
The most advanced and flexible single-vector inducible shRNA available for tightly controlled gene silencing.
Optimized construction of high-quality pools, complete analysis tools, and validated protocols are essential to a successful outcome when screening with pooled lentiviral libraries.
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Technical note describing the SMARTchoice shRNA experimental workflow in suspension cells.
The platform is an innovative system ideally suited for RNAi-mediated studies.
Experimental protocols for gene knockdown using GIPZ lentiviral shRNA.