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RNAi In Vivo |
In vivo RNA interference (RNAi) is crucial for verifying findings from in vitro studies within a complex organism. In vivo verification is performed to understand the effects of complex biological processes, help provide insight into physiological relevance, and to study long-term effects of the RNAi molecule.Unlike in vitro experiments, RNAi in vivo faces distinct challenges due to the complexity of the cellular environment. Achieving meaningful results with small interfering RNA (siRNA) in vivo depends on effective delivery, stability and high-quality of the molecule. Depending on the delivery method and chemical modifications employed, siRNA can remain active in vivo from a few days to several weeks.
Thermo Fisher Scientific offers solutions that overcome many of the inherent challenges of in vivo RNAi work by offering:
- Stabilized siRNA reagents to resist degradation by ribonucleases and minimize immune responses
- HPLC-purified and in vivo-ready RNAi reagents in quantities suitable for animal studies
- Predesigned siRNA for human, mouse, and rat, or custom-designed siRNA to meet your needs
Considerations for siRNA delivery in vivo
When performing in vivo RNAi experiments with synthetic RNA duplexes, it is important to have high-quality material that is well-defined, non-toxic, and sterile, and is compatible with physiological conditions. Thermo Fisher offers in vivo-ready siRNA reagents to help maximize experimental success.
Our portfolio includes three types of siRNA processing and purification for in vivo RNAi research.
- In vivo-ready (IVR)—This process involves standard RNAi oligo synthesis followed by diafiltration to remove salts and solvents to a level <200 µS and sterile filtration, as well as endotoxin testing.
- HPLC—This purity involves standard RNAi oligo synthesis followed by HPLC purification. This step is required for custom siRNAs at large scales and/or that have dyes or other conjugates in order to remove unconjugated material. Does not include extra salt and solvent removal.
- HPLC in vivo (HPLC-IVR)—This purity involves standard RNAi oligo synthesis followed by HPLC purification, diafiltration to remove salts and solvents to a level <200 µS and sterile filtration.
Which in vivo siRNA is right for you?
| Ambion In Vivo siRNA | Stealth RNAi siRNA | Silencer siRNA | |
|---|---|---|---|
| Features | Higher knockdown, prolonged nuclease stability | High knockdown, stabilized | Cost-effective siRNA |
| Relative % knockdown | Higher | High | Moderate |
| Nuclease resistance (without delivery agent) | >48 hours in 90% mouse serum | ~12 hours in 90% mouse serum | <5 min in 90% mouse serum |
| Recommended dosing | 0.05-0.5 mg/kg using Vivofectamine VF232 Reagent | ~1 mg/kg using Invivofectamine 3.0 Reagent | >1 mg/kg using Invivofectamine 3.0 Reagent |
| Target specificity | Highest | High | Moderate |
| Innate immune response | Minimized through chemical modifications | Minimized through chemical modifications | Minimum |
| RNA format | LNA-modified 21-bp duplex with overhangs | Modified 25-bp duplex with no overhangs | Unmodified 21-bp duplex with overhangs |
| Order tool for custom in vivo siRNA designs | Custom Ambion In Vivo siRNA | Custom Stealth RNAi siRNA | Custom Silencer siRNA |
Ambion In Vivo siRNAs
Ambion In Vivo siRNA combines the Silencer Select design algorithm and LNA-modification pattern with additional proprietary modifications for improved serum stability. Ambion In Vivo siRNAs are at least 100x more stable in 90% mouse serum than unmodified siRNAs enabling effective gene knockdown and stability in vivo.
Ambion In Vivo siRNAs, the new standard for in vivo RNAi applications, offer:
- High stability—Proprietary modifications provide resistance against nucleases
- Low immunogenicity—No induction of the interferon response
- Precise and effective silencing—Combining the Silencer Select algorithm and LNA modification pattern leads to potent and sustained gene knockdown with low off-target effects
Effective, targeted knockdown
Ambion In Vivo siRNA targeting Factor VII and PPIB have been successfully delivered by mouse tail vein injection to liver tissue (Figure 1). We demonstrate effective knockdown when measured at the mRNA level.
Figure 1. Ambion in vivo siRNA complexed with Invivofectamine 3.0 Reagent enables targeted knockdown in the liver after a single intravenous injection. Invivofectamine 3.0 Reagent complexed with Ambion In Vivo siRNA targeting mRNA for Factor VII (FVII) or PPIB, injected at doses of 1 mg per kilogram mouse body weight (mg/kg), achieved as much as 85% knockdown of target mRNA levels (knockdown assessed via TaqMan® assay).
Less siRNA required to achieve gene silencing in vivo
Potent, stabilized siRNA combined with effective reagents for in vivo delivery are the key to efficient target gene silencing in animal models. The lower the amount of siRNA required, the lower the chance for adverse effects or off-targets. Complexes of Invivofectamine 3.0 Reagent and Ambion In Vivo siRNA in a range of amounts were introduced via tail vein injection. FVII protein levels in the serum were measured using a chromogenic assay 24 hours after injection (Figure 2). The amount of knockdown is correlated with the amount of siRNA in the complex. The ED50 of Ambion In Vivo siRNA with Invivofectamine 3.0 is 0.1 mg/kg, compared to previous levels of 1.0 mg/kg.
Click image to enlarge
Figure 2. Ambion In Vivo siRNA targeting FVII delivered with Invivofectamine 3.0 Reagent produces dose-response knockdown in liver after a single intravenous injection. Invivofectamine 3.0 Reagent complexed with Ambion In Vivo siRNA targeting FVII was injected at doses ranging from 0.02 to 2 mg/kg. Blood serum was isolated and assayed for FVII protein levels (Biophen® chromogenic assay).
Stealth RNAi siRNA
Stealth RNAi siRNA, a 25-mer blunt-ended RNA duplex, has been chemically modified so only the antisense strand participates in the RNAi pathway, greatly decreasing the potential for off-target effects. The Stealth RNAi siRNA chemical modifications also avoid stimulating a host immune response.
Using Stealth RNAi siRNA for in vivo experiments greatly increased half-life as compared to standard siRNA (Figure 3). This added stability is extremely important for in vivo experiments, given the nuclease-rich environment within an organism.
Click image to enlarge
Figure 3. Stealth RNAi siRNA is stabilized against nuclease degradation in serum. 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 and human serum. Following incubation samples were separated on a Novex 15% TBE-Urea polyacrylamide precast gel and stained with methylene blue.
Fluorescence to verify delivery
To help ensure that the siRNA is delivered to targeted tissues, the following strategies have been used:
- Stealth RNAi siRNA combined with BLOCK-iT Fluorescent control—Stealth RNAi siRNA of interest can be co-transfected with the BLOCK-iT Fluorescent control for tracking of delivery without loss of silencing activity (Figures 4, 5).
- Fluorescently-labeled Stealth RNAi siRNA—Stealth RNAi siRNA can be directly labeled with Alexa Fluor dyes without loss of activity (Figures 4, 5). Order this using our custom in vivo Stealth RNAi ordering tool.
- Biotinylated Stealth RNAi siRNA—RNAi can be biotinylated and combined with either Streptavidin-Alexa Fluor or Streptavidin-Qdot materials (Figures 4, 5). This can be ordered using our custom in vivo Stealth RNAi ordering tool.
Click image to enlarge
Figure 4. The 5’ sense strand of Stealth RNAi siRNA can be modified to allow visualization of uptake without affecting activity. MDA-MB-435 cells were transfected using Lipofectamine RNAiMAX. 24 hours post-transfection, fluorescent uptake was visualized and cells harvested for RNAi analysis.
Click image to enlarge
Figure 5. Effective target silencing is maintained when including Alexa Fluor or biotin conjugations, or when co-transfecting with a labeled control. Cells were transfected with Stealth RNAi siRNA biotin, harvested, and labeled with either Streptavidin-Alexa Fluor 488 or Qdot 655 streptavidin conjugate. The ratio of target gene silencing to GAPDH is maintained when compared to unmodified Stealth RNAi targeting Raf-1.
Vector-based in vivo RNAi
Although employing RNAi vector systems can be slightly more involved than using synthetic siRNA reagents, the flexibility of the vector-based systems is compelling for many researchers. Most RNAi vectors available employ shRNA (short hairpin RNA) vector technology, which typically involves expression of an RNAi effector from a simple stem-loop using a U6 or H1 promoter. More advanced alternatives include a microRNA-derived (miR) scaffold expressing from a Pol II promoter.
RNAi vector delivery methods
Similar to RNAi vectors for in vitro applications, you can use either standard transfection techniques or a viral delivery method to deliver RNAi vectors in vivo.
The delivery of an RNAi expression vector in vivo without using a viral delivery system is fairly similar to delivering plasmid DNA or synthetic dsRNA in vivo. Typically, this would involve complexing the RNAi expression vector with a lipid-based transfection reagent and directly injecting it into the animal. While this may be the easiest approach for delivery of RNAi vectors into animals, it has quite a few limitations, including the inability for systemic delivery and low transfection efficiency. For these reasons, most researchers employing RNAi vectors for in vivo experiments choose to use a viral delivery method.
Regardless of whether one chooses an shRNA or a miR RNAi vector system, the capability for viral delivery is an advantage for many in vivo approaches. Most viral delivery approaches involve either an adenoviral, retroviral (non-lentiviral), or lentiviral technology.
- Adenovirus can be used for transient RNAi expression in either dividing or non-dividing cells.
- Retrovirus can be employed for transient or stable expression but can only be used to transduce dividing cells.
- Lentiviral delivery affords the most options, as it can be used for transient or stable expression in dividing or non-dividing cells; as well as neuronal cells, drug- or growth- arrested cells, or even primary cells (Table 1).
Table 1. Lentiviral delivery offers the most flexibility for delivery of RNAi vectors to a wide variety of cells.
| Viral system | Transient expression | Stable expression | ||||
|---|---|---|---|---|---|---|
| Dividing cells | Nondividing cells | Dividing cells | Neuronal cells | Drug- or growth-arrested cells | Contact-inhibited cells | |
| Adenovirus | ||||||
| Lentivirus | ||||||
| Retrovirus | ||||||
Ambion In Vivo siRNAs
Ambion In Vivo siRNA combines the Silencer Select design algorithm and LNA-modification pattern with additional proprietary modifications for improved serum stability. Ambion In Vivo siRNAs are at least 100x more stable in 90% mouse serum than unmodified siRNAs enabling effective gene knockdown and stability in vivo.
Ambion In Vivo siRNAs, the new standard for in vivo RNAi applications, offer:
- High stability—Proprietary modifications provide resistance against nucleases
- Low immunogenicity—No induction of the interferon response
- Precise and effective silencing—Combining the Silencer Select algorithm and LNA modification pattern leads to potent and sustained gene knockdown with low off-target effects
Effective, targeted knockdown
Ambion In Vivo siRNA targeting Factor VII and PPIB have been successfully delivered by mouse tail vein injection to liver tissue (Figure 1). We demonstrate effective knockdown when measured at the mRNA level.
Figure 1. Ambion in vivo siRNA complexed with Invivofectamine 3.0 Reagent enables targeted knockdown in the liver after a single intravenous injection. Invivofectamine 3.0 Reagent complexed with Ambion In Vivo siRNA targeting mRNA for Factor VII (FVII) or PPIB, injected at doses of 1 mg per kilogram mouse body weight (mg/kg), achieved as much as 85% knockdown of target mRNA levels (knockdown assessed via TaqMan® assay).
Less siRNA required to achieve gene silencing in vivo
Potent, stabilized siRNA combined with effective reagents for in vivo delivery are the key to efficient target gene silencing in animal models. The lower the amount of siRNA required, the lower the chance for adverse effects or off-targets. Complexes of Invivofectamine 3.0 Reagent and Ambion In Vivo siRNA in a range of amounts were introduced via tail vein injection. FVII protein levels in the serum were measured using a chromogenic assay 24 hours after injection (Figure 2). The amount of knockdown is correlated with the amount of siRNA in the complex. The ED50 of Ambion In Vivo siRNA with Invivofectamine 3.0 is 0.1 mg/kg, compared to previous levels of 1.0 mg/kg.
Click image to enlarge
Figure 2. Ambion In Vivo siRNA targeting FVII delivered with Invivofectamine 3.0 Reagent produces dose-response knockdown in liver after a single intravenous injection. Invivofectamine 3.0 Reagent complexed with Ambion In Vivo siRNA targeting FVII was injected at doses ranging from 0.02 to 2 mg/kg. Blood serum was isolated and assayed for FVII protein levels (Biophen® chromogenic assay).
Stealth RNAi siRNA
Stealth RNAi siRNA, a 25-mer blunt-ended RNA duplex, has been chemically modified so only the antisense strand participates in the RNAi pathway, greatly decreasing the potential for off-target effects. The Stealth RNAi siRNA chemical modifications also avoid stimulating a host immune response.
Using Stealth RNAi siRNA for in vivo experiments greatly increased half-life as compared to standard siRNA (Figure 3). This added stability is extremely important for in vivo experiments, given the nuclease-rich environment within an organism.
Click image to enlarge
Figure 3. Stealth RNAi siRNA is stabilized against nuclease degradation in serum. 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 and human serum. Following incubation samples were separated on a Novex 15% TBE-Urea polyacrylamide precast gel and stained with methylene blue.
Fluorescence to verify delivery
To help ensure that the siRNA is delivered to targeted tissues, the following strategies have been used:
- Stealth RNAi siRNA combined with BLOCK-iT Fluorescent control—Stealth RNAi siRNA of interest can be co-transfected with the BLOCK-iT Fluorescent control for tracking of delivery without loss of silencing activity (Figures 4, 5).
- Fluorescently-labeled Stealth RNAi siRNA—Stealth RNAi siRNA can be directly labeled with Alexa Fluor dyes without loss of activity (Figures 4, 5). Order this using our custom in vivo Stealth RNAi ordering tool.
- Biotinylated Stealth RNAi siRNA—RNAi can be biotinylated and combined with either Streptavidin-Alexa Fluor or Streptavidin-Qdot materials (Figures 4, 5). This can be ordered using our custom in vivo Stealth RNAi ordering tool.
Click image to enlarge
Figure 4. The 5’ sense strand of Stealth RNAi siRNA can be modified to allow visualization of uptake without affecting activity. MDA-MB-435 cells were transfected using Lipofectamine RNAiMAX. 24 hours post-transfection, fluorescent uptake was visualized and cells harvested for RNAi analysis.
Click image to enlarge
Figure 5. Effective target silencing is maintained when including Alexa Fluor or biotin conjugations, or when co-transfecting with a labeled control. Cells were transfected with Stealth RNAi siRNA biotin, harvested, and labeled with either Streptavidin-Alexa Fluor 488 or Qdot 655 streptavidin conjugate. The ratio of target gene silencing to GAPDH is maintained when compared to unmodified Stealth RNAi targeting Raf-1.
Vector-based in vivo RNAi
Although employing RNAi vector systems can be slightly more involved than using synthetic siRNA reagents, the flexibility of the vector-based systems is compelling for many researchers. Most RNAi vectors available employ shRNA (short hairpin RNA) vector technology, which typically involves expression of an RNAi effector from a simple stem-loop using a U6 or H1 promoter. More advanced alternatives include a microRNA-derived (miR) scaffold expressing from a Pol II promoter.
RNAi vector delivery methods
Similar to RNAi vectors for in vitro applications, you can use either standard transfection techniques or a viral delivery method to deliver RNAi vectors in vivo.
The delivery of an RNAi expression vector in vivo without using a viral delivery system is fairly similar to delivering plasmid DNA or synthetic dsRNA in vivo. Typically, this would involve complexing the RNAi expression vector with a lipid-based transfection reagent and directly injecting it into the animal. While this may be the easiest approach for delivery of RNAi vectors into animals, it has quite a few limitations, including the inability for systemic delivery and low transfection efficiency. For these reasons, most researchers employing RNAi vectors for in vivo experiments choose to use a viral delivery method.
Regardless of whether one chooses an shRNA or a miR RNAi vector system, the capability for viral delivery is an advantage for many in vivo approaches. Most viral delivery approaches involve either an adenoviral, retroviral (non-lentiviral), or lentiviral technology.
- Adenovirus can be used for transient RNAi expression in either dividing or non-dividing cells.
- Retrovirus can be employed for transient or stable expression but can only be used to transduce dividing cells.
- Lentiviral delivery affords the most options, as it can be used for transient or stable expression in dividing or non-dividing cells; as well as neuronal cells, drug- or growth- arrested cells, or even primary cells (Table 1).
Table 1. Lentiviral delivery offers the most flexibility for delivery of RNAi vectors to a wide variety of cells.
| Viral system | Transient expression | Stable expression | ||||
|---|---|---|---|---|---|---|
| Dividing cells | Nondividing cells | Dividing cells | Neuronal cells | Drug- or growth-arrested cells | Contact-inhibited cells | |
| Adenovirus | ||||||
| Lentivirus | ||||||
| Retrovirus | ||||||
Order in vivo siRNA
In vivo ready- Silencer and Silencer Select siRNA controls
Stylesheet for Classic Wide Template adjustments
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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
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