by Caitlin Smith
RNA interference (RNAi) – the process of disrupting gene expression such that the gene’s corresponding protein is reduced or “knocked down” – has rapidly become an important gene expression tool since its introduction several years ago. With the widespread use of this gene silencing technique came a quick evolution of RNAi tools, such as small interfering RNA (siRNA), also known as small inhibitory RNA. Today, these small double-stranded molecules of RNA are more high-tech than their ancestors. Researchers have tweaked them to make siRNAs more effective. “We’ve added chemical modifications that improve their stability and potency, and reduce the immunogenic response and off-target effects,” says Xavier de Mollrat du Jeu, senior staff scientist at Life Technologies. “Using chemical modifications is key to reducing the unwanted complications encountered in vivo, like serum, nucleases, degradations, and immune response.”
Designing siRNAs has also been well-studied, with established design guidelines and algorithms. “Modern design algorithms make siRNAs a very reliable biological tool and address potential off-target effects,” says Jörg Dennig, global product manager for siRNA at Qiagen. Once you know what gene you want to silence, you can use one of many good online design tools on vendor websites. Some vendors even offer an efficacy guarantee on the 3 or 4 siRNAs designed by their online design tool.
Once you know the siRNAs you want, what’s the best way to prepare them? “I would start by asking the following questions,” says Louise Carr Baskin, product manager in siRNA and transfection for Genomics at Thermo Fisher Scientific. “Do you want siRNA that’s ready-to-use, or are you willing to carry out some preparation steps in return for a renewable resource? Will you need to utilize modified RNA bases for stability or specificity, or is an unmodified siRNA suitable for your needs? Will you be carrying out long-term silencing studies, or is transient silencing (4-6 days) suitable for your experiment?” As you ponder these questions, consider the pros and cons of the four main methods of preparing siRNAs: chemical synthesis, in vitro transcription, PCR expression cassettes, and siRNA expression vectors.
The use of siRNAs prepared by chemical synthesis is widespread, and for many reasons. This method results in siRNAs of high quality, produced in a controlled fashion according to the exact sequence you require, even including chemical modifications. “Their reproducible purity and quality is higher than those produced by enzyme-driven reactions,” says Baskin. “[Also advantageous] is the ability to incorporate modifications to the backbone and/or RNA bases to improve siRNA target specificity, stability in the presence of nucleases, and improve strand loading properties.” Drawbacks to chemically synthesized siRNAs include higher cost, and only transient gene silencing, not long-term RNAi experiments.
The broad availability of synthetic siRNAs make it easier to find exactly what you need. "Even if someone is working with an unusual model organism or has a highly specialized siRNA design request, customized siRNA synthesis is a fast, high-confidence method for targeted gene silencing,” says Baskin. Thermo Fisher Scientific’s ON-TARGETplus specificity-enhanced product line includes 4 siRNAs per gene, as does their siGENOME collection. Transfection reagents help to introduce the siRNAs into cells, such as Roche’s X-tremeGENE siRNA Transfection Reagent, and Life Technologies’ Lipofectamine RNA Max. Life Technologies recently released a new transfect reagent, Invivofectamine 2.0, for introducing siRNAs into whole animals. Qiagen’s FlexiTube siRNA Premixes combine siRNAs and transfection reagent in a stabilized complex that keeps for up to a month.
in vitro transcription
The in vitro transcription method of preparing siRNAs involves generating a long double-stranded RNA molecule, and then cleaving it enzymatically into siRNA duplexes. There are a lot of steps involved, so most labs wouldn’t want to use this method to generate a large collection of siRNAs. The preparatory steps include generating the DNA template, transcribing two strands, cleaving into siRNAs, and selecting the right region for DNA template amplification. Another caveat of this method is that the rational design rules for functional siRNA (incorporated into online design tools for synthesized siRNAs) cannot be followed in generating them. As a result, in vitro transcription may generate many nonspecific oligomers. Furthermore, “the enzymatic steps have varying fidelity that results in dsRNA of varying length which can be highly toxic to cells,” says Baskin. “Therefore purification of the small siRNA is required, which can compromise total siRNA yield.”
Generally, preparing siRNAs by in vitro transcription is suitable for making only a few siRNAs quickly. “A major advantage of the in vitro transcription approach is that there is a higher probability that the pool of siRNAs will contain effective molecules (those with good accessibility to their target mRNA sequences),“ says Lauren Buck, associate marketing manager at Roche Applied Science. Though they require more work with less assurance of results, these siRNAs are less expensive than chemically synthesized siRNAs.
PCR expression cassettes
Another quick and cost-effective method of preparing siRNAs is the use of PCR expression cassettes. According to Buck, "this method has fewer steps and is a good choice for siRNA target site screening because multiple cassettes can be generated relatively easily.“ PCR expression cassettes are essentially “short hairpin RNA (shRNA) in DNA form that can be amplified with primers containing a promoter and terminator, respectively,” says Baskin. “This PCR cassette is then transfected into cells and the shRNA is expressed.” Drawbacks to this method include potential difficulties with PCR amplification due to the hairpin secondary structure, and inefficient DNA transfection into cells due to the cassette’s absence of a selectable marker. “This method has some flexibility, however, because primers with different promoters can be generated,” Baskin adds.
siRNA expression vectors
Like expression cassettes, siRNA expression vectors also rely on the expression of shRNA that will go on to produce siRNA. However, unlike the above preparation approaches, siRNA expression methods also result a longer-term siRNA production system, not simply a one-time exercise. “Although there is more significant hands-on time than other methods, the resulting plasmid represents a renewable resource,” notes Baskin. After you design the shRNA and clone it into a plasmid, you must also verify the sequence of the resulting vector. The main disadvantage of shRNA expression vectors is that cloning and sequence verification can be time consuming and expensive,” says Baskin. “The key advantage of expression-based RNAi is the ability to use a vector with a selectable marker to select for cells that are transfected with the plasmid and to carry out long-term silencing studies. In addition, if the vector contains elements for viral packaging, it can be packaged into viral particles that will allow for transduction into many different types of cells.” Baskin says that many vendors sell shRNA expression vectors as bacterial glycerol stocks from which plasmid preparation can be carried out.
Using siRNA expression vectors to perform RNAi experiments in cultured cells using selectable markers or inducible expression systems is particularly advantageous. “Researchers can use siRNA expression vectors that allow the efficient generation of stable cell lines via resistance markers,” says Dennig. “Qiagen’s SureSilencing shPlasmids include different selection markers, hygromycin, puromycin, or neomycin resistance, or alternatively with GFP. This allows researchers to select the siRNA expression vectors with the resistance gene of their choice for the generation of stable cell lines for gene silencing experiments.” Buck agrees that “vector-based siRNA is generally preferable to synthetic siRNA when it is important to achieve physiologically relevant results,” she says. “Vectors allow establishment of stable cell lines and inducible expression systems. However, this method requires significant preparation which can mean higher costs and time delays.”
Though the methods to prepare siRNAs differ, a considered choice of the proper approach for your lab should yield siRNAs in abundance.
The graph at the top of this article is data from Thermo Fisher Scientific's Accell siRNA Reagents.