siRNA Synthesis Services

Companies are not only responding to the popularity of siRNAs with their services—designing siRNAs can be tricky. “The two challenges in siRNA design are potency and specificity,” says Bettina Haedrich, global product manager of gene silencing at Qiagen. “siRNAs should be potent, providing efficient target knockdown at low siRNA concentrations. siRNA should also be specific with minimal risk of off-target effects.” Jutta Huber, manager of business development at MWG Biotech, agrees: “Regarding design, the most critical issues are off target effects.” Undesirable off-target effects can occur after the siRNA duplex unwinds, when one of the strands binds to a complementary, but nontargeted, sequence.

Another challenge in designing and synthesizing siRNAs is finding the right target sites. “While current design algorithms are advanced, not all gene targets are equal,” explains Lynette Brown, product manager at Integrated DNA Technologies (IDT). “A subset of genes are ‘hard targets’ (maybe 15-20% of all genes) and finding good siRNA sites can be a real challenge. Why are these genes hard targets? No one knows for sure, but we speculate that there may be issues with mRNA and its secondary and/or tertiary structure which interferes with siRNA guide strand binding. Also, there may be issues with mRNA localization which further limits access of the siRNA and RISC to the target.” In learning about how different companies tackle these obstacles, you may discover which siRNA synthesis service is right for you.

Qiagen offers its HPP (High-Performance Purity) grade siRNA as an economical yet pure option. “This is the optimal siRNA purification grade for efficient gene silencing at an affordable cost,” says Haedrich. It is synthesized using proprietary synthesis and purification processes, yielding siRNA that is greater than 90% pure. Qiagen claims that for many applications, there is no need for additional purification steps (such as HPLC or PAGE) of their siRNAs. Each individual siRNA type then undergoes stringent tests for quality, including MALDI-TOF mass spectrometry analysis. If this level of purity is not high enough for your applications, they do custom synthesis of siRNAs that are subject to HPLC purification. You can also buy predesigned siRNAs for every human, mouse, and rat gene, as well as validated siRNAs, which “offer guaranteed performance because they have been tested by real-time RT-PCR and shown to provide at least 70% target gene knockdown,” says Haedrich.

For sets of siRNAs, Qiagen offers FlexiPlate siRNA. Through their website, you can search for your siRNAs of interest, specify the scale of siRNA that you need, and arrange all the siRNAs, including controls, in a 96-well plate. “FlexiPlate siRNA enables maximum flexibility for pathway analysis or follow-up screens,” says Haedrich.

Qiagen tries to reduce the risk of off-target effects with its design tool. “HP OnGuard siRNA Design uses advanced neural network technology to select potent siRNA,” says Haedrich. “siRNA design is checked for homology to all other sequences of the genome using an up-to-date, nonredundant sequence database and a proprietary homology analysis tool to ensure specificity. Innovative design features, such as 3' UTR/seed region analysis and interferon motif avoidance, use advances in the knowledge of the RNAi mechanism to reduce the risk of off-target effects.”

MWG Biotech’s siMAX siRNAs are available with or without HPLC purification, and in three scales (0.05 mmol, 0.2 mmol, or 1.0 mmol). Predesigned siRNAs are also available for human, mouse, and rat cells.

Falcetti explains a possible advantage of the design of Invitrogen’s Stealth siRNAs—they can mitigate off-target effects: “When designing siRNAs there are two strands that can potentially participate in mRNA knockdown. This could lead to inhibition of unintended targeting genes. With Stealth RNAi only one strand participates in knockdown, reducing the potential for such off-target effects.”

IDT has developed another modified type of siRNA called Dicector Dicer-substrate siRNAs (DsiRNAs). When used at low concentrations (10 nM or less), these are more potent than conventional siRNAs. “DsiRNAs are chemically synthesized 27-mer duplex RNAs,” says Lynette Brown, product manager at IDT. “They are processed by Dicer into 21-mer siRNAs and designed so that cleavage results in a single, desired product. New design rules specific to this process have been developed and are only available from IDT.” DsiRNAs are purified by HPLC to greater than 90% duplex purity as verified by analytical HPLC and ESI-mass spectrometry.

Dicector Dicer-substrate siRNAs are available as custom designed or predesigned, as well as in kit format. IDT’s TriFECTa kit includes three pre-designed gene specific duplexes and three control duplexes. “Each kit is guaranteed to provide greater than 70% knockdown with at least two of the three DsiRNA duplexes when used at 10 nM concentration and assayed by quantitative RT-PCR,” says Brown. “For the guarantee to be valid, the fluorescent transfection control duplex must indicate that greater than 90% of the cells have been transfected, and the HPRT positive control works with the expected efficiency (which is greater than 90% knockdown at 10 nM).” Both predesigned and custom designed siRNAs can benefit from Invitrogen’s free online design tools.

GenScript’s siRNAs are purified by PAGE, and also come with a guarantee: “By using the GenScript siRNA design center and custom siRNA, there is a 95% chance that you will find a potent siRNA target (70% inhibition) if you test 3 siRNA constructs for a gene,” says Sharma. “The most common problem for siRNA failure is the transfection efficiency. GenScript has developed a GFP marker vector to address that issue. However, Genscript does not guarantee that the recommended siRNA constructs will work because of uncertainty in the transfection experiments.”

One way to avoid the uncertainty of short-term transfection experiments is to use stably transfected cell lines instead. Transfection with siRNA to create stable cell lines is a specialty of VicGene. “Vicgene supplies siRNA in linearized DNA forms with antibiotics selection markers to facilitate establishing stably transfected cell lines in a very short time,” says Richard Liu, VicGene technical support professional. “The constructs also contain special DNA sequences to enhance the integration of the entire constructs into host cell chromosomes at a high frequency.” VicGene offers a measure of guarantee for knockdown results, as well: “All of our siRNA products have been tested, and are guaranteed to give more than 75% gene silencing efficiency.”

Whether you intend to silence your gene of interest in short-term transfection experiments, stably transfected cell lines, or through in vivo transfection, the variety of siRNA synthesis services and styles available will surely satisfy your experimental needs. If not, talk to them—most custom services are willing to work with customers to find the best siRNA for their particular experiments.