Invitrogen Stealth RNAi siRNA uses next-generation RNAi chemistry that provides higher specificity and increased stability in serum and cell culture than standard siRNA. This chemistry produces cleaner results while eliminating unwanted off-target effects providing:

Stealth RNAi siRNA is 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. To order your gene-specific Stealth RNAi reagents, use the search tool below.

Order your Stealth RNAi siRNA positive and negative controls

Stealth RNAi siRNA positive controls
Stealth RNAi siRNA negative controls

Benefits of Stealth siRNA next-generation chemistry

Stealth RNAi siRNA provides effective knockdown to ensure silencing of the target gene. Figure 1 demonstrates comparable silencing between Stealth RNAi and an unmodified siRNA Stealth RNAi provides a functional guaranteed 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%. Learn more about our functionality guarantees.

Bar graph depicting comparable p53 gene silencing of standard and Stealth RNAi siRNA
Figure 1. Reduction in expression of p53 in A549 cells by siRNA and Stealth RNAi siRNA delivered using Invitrogen Lipofectamine 2000. Corresponding controls consisted of chemistry matched, scrambled sequences with a similar base-pair composition. Reduction in p53 expression is presented as fold change in expression of p53 normalized to GAPDH relative to the corresponding control measured by quantitative real-time PCR.

Off-target effects occur when an siRNA has sufficient homology to an untargeted gene, thereby silencing it along with the intended target. Stealth RNAi can eliminate 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 strand of an unmodified siRNA can enter the RNAi pathway. But Stealth RNAi modifications only allow the antisense strand to efficiently enter the RNAi pathway. This modification eliminates concerns about sense strand off-target effects.

Bar graph comparing specificity for target gene between Stealth RNAi and standard siRNA.

Figure 2. Stealth RNAi siRNA exhibits increased specificity for targets.

The Stealth RNAi 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, avoiding the need to develop and test different molecules for animal studies and cell culture work.

Stealth RNAi siRNA stability is improved for up to 72 hours compared to standard siRNA using denaturing PAGE
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.

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 eliminates the induction of the PKR/interferon response pathway, ensuring cleaner results in RNAi experiments (Figure 4). Using Stealth RNAi enables potent gene knockdown without the risk of activating the cell’s stress responses that make results difficult to interpret.

Bar graph of siRNAs and controls illustrating minimal interferon response of Stealth RNAi siRNAs relative to standard siRNA counterparts
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.

Storage and stability of siRNA reagents:

Store as a dry pellet at –20°C until ready to use. Once resuspended, store at –20°C and avoid contact with RNAses. Store at –20°C for at least a year.

Resuspending siRNA

Resuspend siRNA or Stealth RNAi duplexes in DEPC-treated water according to the chart in order to make a 20 µM solution. The RNA oligo was dried down from a buffered solution. Resuspension to 20 µM will reconstitute the buffer to 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA.

Delivered Quantity/PurityResuspension Protocol
20 nmole desaltedResuspend the 20 nmole yield in 1000 µl RNase-free water in order to make a 20 µM solution.
80 nmole desaltedResuspend the 80 nmole yield in 800 µl RNase-free water to make a 100 µM solution. Dilute 1:5 to create a 20 µM working stock.
1.0 µmole desaltedResuspend the 1 µmole yield in 1 ml RNase-free water to make a 1mM solution. Dilute 1:50 to create a 20 µM working stock.

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.
Effective
knockdown

Stealth RNAi siRNA provides effective knockdown to ensure silencing of the target gene. Figure 1 demonstrates comparable silencing between Stealth RNAi and an unmodified siRNA Stealth RNAi provides a functional guaranteed 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%. Learn more about our functionality guarantees.

Bar graph depicting comparable p53 gene silencing of standard and Stealth RNAi siRNA
Figure 1. Reduction in expression of p53 in A549 cells by siRNA and Stealth RNAi siRNA delivered using Invitrogen Lipofectamine 2000. Corresponding controls consisted of chemistry matched, scrambled sequences with a similar base-pair composition. Reduction in p53 expression is presented as fold change in expression of p53 normalized to GAPDH relative to the corresponding control measured by quantitative real-time PCR.
Higher
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 can eliminate 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 strand of an unmodified siRNA can enter the RNAi pathway. But Stealth RNAi modifications only allow the antisense strand to efficiently enter the RNAi pathway. This modification eliminates concerns about sense strand off-target effects.

Bar graph comparing specificity for target gene between Stealth RNAi and standard siRNA.

Figure 2. Stealth RNAi siRNA exhibits increased specificity for targets.

Greater
stability

The Stealth RNAi 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, avoiding the need to develop and test different molecules for animal studies and cell culture work.

Stealth RNAi siRNA stability is improved for up to 72 hours compared to standard siRNA using denaturing PAGE
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.
Less 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 eliminates the induction of the PKR/interferon response pathway, ensuring cleaner results in RNAi experiments (Figure 4). Using Stealth RNAi enables potent gene knockdown without the risk of activating the cell’s stress responses that make results difficult to interpret.

Bar graph of siRNAs and controls illustrating minimal interferon response of Stealth RNAi siRNAs relative to standard siRNA counterparts
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.
Handling &
resuspension

Storage and stability of siRNA reagents:

Store as a dry pellet at –20°C until ready to use. Once resuspended, store at –20°C and avoid contact with RNAses. Store at –20°C for at least a year.

Resuspending siRNA

Resuspend siRNA or Stealth RNAi duplexes in DEPC-treated water according to the chart in order to make a 20 µM solution. The RNA oligo was dried down from a buffered solution. Resuspension to 20 µM will reconstitute the buffer to 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA.

Delivered Quantity/PurityResuspension Protocol
20 nmole desaltedResuspend the 20 nmole yield in 1000 µl RNase-free water in order to make a 20 µM solution.
80 nmole desaltedResuspend the 80 nmole yield in 800 µl RNase-free water to make a 100 µM solution. Dilute 1:5 to create a 20 µM working stock.
1.0 µmole desaltedResuspend the 1 µmole yield in 1 ml RNase-free water to make a 1mM solution. Dilute 1:50 to create a 20 µM working stock.

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.
Resources
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