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Stealth RNAi siRNAs |
Invitrogen Stealth RNAi siRNA uses RNAi chemistry that enables higher specificity and increased stability in serum and cell culture than standard siRNA. This chemistry typically produces cleaner results and helps minimize unwanted off-target effects, offering:
Stealth RNAi siRNA are 25-mer duplexes manufactured with the strictest quality control standards. Each single-stranded RNA oligo is analyzed by mass spec and then annealed to deliver the specified amount of duplex. Every Stealth RNAi siRNA is backed by an industry-standard guarantee.
Benefits of Stealth RNAi
Click on the tabs below to view data on particular benefits of stealth siRNAs.
Effective knockdown
Stealth RNAi siRNA enables effective knockdown to help ensure silencing of the target gene. Figure 1 demonstrates comparable silencing between Stealth RNAi and an unmodified siRNA. Stealth RNAi offers a functional guarantee that at least 2 out of the 3 reagents per gene will result in at least 70% transcript knockdown, given that the transfection efficiency in your experiment is at least 80%. Visit our siRNA guarantee page for more information.
Figure 1. Reduction in expression of p53 in A549 cells by Stealth RNAi siRNA delivered using Lipofectamine RNAiMAX Transfection Reagent. Reduction in p53 expression is measured by quantitative real-time PCR and presented as a fold change in expression of p53 normalized to GAPDH relative to the cells only control.
High specificity
Off-target effects occur when an siRNA has sufficient homology to an untargeted gene, thereby silencing it along with the intended target. Stealth RNAi siRNA can help minimize sense strand–mediated off-target effects that can be problematic with traditional siRNA, even at low concentrations. These problems can arise because both the sense and antisense strands of an unmodified siRNA can enter the RNAi pathway. But Stealth RNAi siRNA modifications only allow the antisense strand to efficiently enter the RNAi pathway. This modification alleviates concerns about sense strand off-target effects.
Figure 2. Stealth RNAi siRNA exhibits increased specificity for targets.
Optimal stability
The Stealth RNAi siRNA modifications also increase stability when compared to traditional, unmodified siRNA. Traditional siRNAs are degraded over time in serum containing nucleases, making them undesirable for use in animals.
However, Stealth RNAi siRNA remains stable for up to 72 hours (Figure 3), making it a better choice for projects that involve work with animal models. This flexibility can save weeks of time, reducing the need to develop and test different molecules for animal studies and cell culture work.
Figure 3. Stealth RNAi siRNA is more stable in serum than standard siRNA. Unmodified 21-mer dsRNA sequence (left panel) and corresponding Stealth RNAi siRNA sequence (right panel) at 0, 4, 8, 24, 48, and 72 hours following incubation in 10% mouse serum. Following incubation samples were separated on an Invitrogen Novex 15% TBE-Urea polyacrylamide precast gel.
Minimal cellular toxicity
Studies with standard siRNA have documented that unmodified siRNAs can induce cellular stress response pathways, such as the interferon response, that can result in growth inhibition and cellular toxicity. This makes it difficult to assess whether observed cellular phenotypes are due to non-specific stress responses, or to loss-of-function of a targeted gene. Stealth RNAi siRNA doesn’t induce the PKR/interferon response pathway, enabling cleaner results in RNAi experiments (Figure 4). Using Stealth RNAi siRNA enables potent gene knockdown without the risk of activating the cell’s stress responses that make results difficult to interpret.
Figure 4. Stealth RNAi siRNA avoids induction of interferon response genes. An siRNA sequence induced multiple interferon genes following delivery into A549 cells. A Stealth RNAi siRNA version of the same siRNA sequence did not alter expression of interferon response genes.
Effective knockdown
Stealth RNAi siRNA enables effective knockdown to help ensure silencing of the target gene. Figure 1 demonstrates comparable silencing between Stealth RNAi and an unmodified siRNA. Stealth RNAi offers a functional guarantee that at least 2 out of the 3 reagents per gene will result in at least 70% transcript knockdown, given that the transfection efficiency in your experiment is at least 80%. Visit our siRNA guarantee page for more information.
Figure 1. Reduction in expression of p53 in A549 cells by Stealth RNAi siRNA delivered using Lipofectamine RNAiMAX Transfection Reagent. Reduction in p53 expression is measured by quantitative real-time PCR and presented as a fold change in expression of p53 normalized to GAPDH relative to the cells only control.
High specificity
Off-target effects occur when an siRNA has sufficient homology to an untargeted gene, thereby silencing it along with the intended target. Stealth RNAi siRNA can help minimize sense strand–mediated off-target effects that can be problematic with traditional siRNA, even at low concentrations. These problems can arise because both the sense and antisense strands of an unmodified siRNA can enter the RNAi pathway. But Stealth RNAi siRNA modifications only allow the antisense strand to efficiently enter the RNAi pathway. This modification alleviates concerns about sense strand off-target effects.
Figure 2. Stealth RNAi siRNA exhibits increased specificity for targets.
Optimal stability
The Stealth RNAi siRNA modifications also increase stability when compared to traditional, unmodified siRNA. Traditional siRNAs are degraded over time in serum containing nucleases, making them undesirable for use in animals.
However, Stealth RNAi siRNA remains stable for up to 72 hours (Figure 3), making it a better choice for projects that involve work with animal models. This flexibility can save weeks of time, reducing the need to develop and test different molecules for animal studies and cell culture work.
Figure 3. Stealth RNAi siRNA is more stable in serum than standard siRNA. Unmodified 21-mer dsRNA sequence (left panel) and corresponding Stealth RNAi siRNA sequence (right panel) at 0, 4, 8, 24, 48, and 72 hours following incubation in 10% mouse serum. Following incubation samples were separated on an Invitrogen Novex 15% TBE-Urea polyacrylamide precast gel.
Minimal cellular toxicity
Studies with standard siRNA have documented that unmodified siRNAs can induce cellular stress response pathways, such as the interferon response, that can result in growth inhibition and cellular toxicity. This makes it difficult to assess whether observed cellular phenotypes are due to non-specific stress responses, or to loss-of-function of a targeted gene. Stealth RNAi siRNA doesn’t induce the PKR/interferon response pathway, enabling cleaner results in RNAi experiments (Figure 4). Using Stealth RNAi siRNA enables potent gene knockdown without the risk of activating the cell’s stress responses that make results difficult to interpret.
Figure 4. Stealth RNAi siRNA avoids induction of interferon response genes. An siRNA sequence induced multiple interferon genes following delivery into A549 cells. A Stealth RNAi siRNA version of the same siRNA sequence did not alter expression of interferon response genes.
Stealth RNAi and siRNA transfection concentrations
The transfection concentration of a Stealth RNAi siRNA or siRNA duplex is determined by dividing the molar amount used by the final volume of the transfection (i.e., starting medium volume plus transfection mixture volume). With a potent siRNA, we typically transfect 0.5–5 pmol by adding 100 µL transfection mix to 500 µL medium (0.8–8 nM final concentration) per well in a 24-well plate. To scale up, increase the amount of siRNA to keep the concentration the same. We recommend using the minimum amount of siRNA that will knockdown your gene to avoid any non-specific or off-target effects.
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Questions?
Technical inquires:
Our Technical Application Scientists are available to help assist you at techsupport@thermofisher.com
Ordering & Order Status inquires:
If you have questions about pre-designed RNAi orders and order status, please contact us at genomicorders@thermofisher.com
If you have any questions about Custom RNAi orders and order status, please contact us at RNAiSupport@thermofisher.com
For Research Use Only. Not for use in diagnostic procedures.
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