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We offer solutions that overcome many of the inherent challenges of in vivo RNAi work by providing:
In vivo RNAi experiments are more challenging than their invitro counterparts due to the demands of the cellular environment. These added challenges necessitate the use of the highest quality materials to obtain meaningful results. Thermo Fisher Scientific offers a complete solution for successful in vivo RNAi experiments, including the highest quality chemically modified siRNA.
Highest knockdown, prolonged nuclease stability | High knockdown, stabilized | Cost-effective siRNA | |
---|---|---|---|
Ambion In Vivo siRNA | Stealth RNAi siRNA | Silencer siRNA | |
Relative % knockdown | Highest | 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 with Invivofectamine 3.0 | ~1 mg/kg | ~1 mg/kg | >1 mg/kg |
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 |
When performing in vivo RNAi experiments with synthetic RNA duplexes, it is very important to have well-defined, non-toxic, and sterile starting material, compatible with physiological conditions. Thermo Fisher Scientific offers in vivo-ready siRNA reagents to maximize experimental success.
Our portfolio includes three types of siRNA processing and purification for in vivo RNAi research.
Ambion In Vivo siRNA molecules are chemically modified, 21-mer, double-stranded siRNAs that are recognized by the RNA-induced silencing complex (RISC) to mediate inhibition of a target gene. Proprietary chemical modifications allow Ambion In Vivo siRNAs to overcome many in vivo–specific obstacles, ensuring their effectiveness and stability in vivo. Ambion In Vivo siRNAs are at least 100x more stable in 90% mouse serum than unmodified siRNAs.
Ambion In Vivo siRNAs, the new standard for in vivo RNAi applications, offer:
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).
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.
Figure 2. Ambion In Vivo siRNA targeting FVII delivered with Invivofectamine 3.0 Reagent produce 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, a chemically modified 25-mer blunt-ended RNA duplex, has been chemically altered so only the antisense strand participates in the RNAi pathway, greatly decreasing the potential for off-target effects. Additionally, the chemical modification also allows Stealth RNAi siRNA to 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.
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. Following incubation samples were separated on a Novex 15% TBE-Urea polyacrylamide precast gel and stained with methylene blue.
To help ensure that the siRNA is delivered to targeted tissues, the following strategies have been used:
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 (human breast carcinoma) were transfected using Lipofectamine RNAiMAX. 24 hours post-transfection, fluorescent uptake was visualized and cells harvested for RNAi analysis.
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
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.
Learn more about lentiviral Pol II miR RNAi expression systems
The BLOCK-iT Pol II miR RNAi Expression Vectors offer significant advantages over shRNA vector technology. They retain the ability to achieve stable expression use of viral delivery, but also include capabilities for tissue-specific expression and multiple target knockdown from the same transcript.
The HiPerform Lentiviral Pol II miR RNAi Expression System with EmGFP(Figure 6) contains an mRNA stabilizing sequence (WPRE) and a nuclear import sequence (cPPT) which have generated up to 5-fold higher virus titers and EmGFP expression levels in many cell lines. Additionally, MultiSite technology allows you to express the EmGFP/miR RNAi cassette from CMV, EF-1a, or your own tissue-specific or other in vivo-appropriate promoter.
Figure 6. pLenti6/V5-DEST vector. The BLOCK-iT HiPerform Lentiviral POL II miR RNAi Expression System with EmGFP. The pLenti6.4/CMVor Ef-1a/V5-M5--GW/EmGFP-miR vector is driven by the CMV promotor, has the blasticidin resistance marker, and is available with co-cistronic EmGFP expression as a reporter.
Learn more about lentiviral Pol II miR RNAi expression systems
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 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 efficiencies. 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
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 |
For answers to additional questions, please refer to the Thermo Fisher Scientific technical support FAQ database or contact RNAiSupport@thermofisher.com to have a representative assist you.
A. Pre-designed Stealth RNAi and Ambion In Vivo siRNA can be ordered directly from our pre-designed siRNA search interface to target human, mouse, or rat genes.
Custom siRNA designs in vivo experiments can be ordered from two interfaced, depending on the product format being requested: Custom Ambion In Vivo siRNA is ordered from the GeneAssist Custom siRNA Builder, while Stealth RNAi is ordered using the BLOCK-iT RNAi Express for in vivo synthetics .
A. Chemically modified siRNA duplexes, such as Ambion In Vivo siRNA and Stealth RNAi, have a number of advantages over standard RNAi duplexes, including the minimization of off-target effects, enhanced stability, and reduced toxicity. For these reasons, chemically modified RNAi duplexes are recommended for in vivo RNAi experiments.
A. We have demonstrated that labeling Stealth RNAi duplexes does not hamper their knockdown potency. An alternative approach is to mix unlabeled duplexes with labeled control duplexes; this method is more commonly used with in vivo RNAi and allows progression to clinical research unhindered by questions about the possible effects of a dye.
A. Our mirVana Mimics and Inhibitors are available as pre-designed against mirBase v22 content in 250 nmol amounts, in vivo-ready and HPLC purified. Custom designs and other synthesis options can be ordered from our GeneAssist miRNA ordering tool.
A. Production of in vivo RNAi duplexes begins with standard synthesis of RNAi oligos using high-quality starting materials. The RNA oligos are then duplexed and desalted. At this point, the researcher can also request HPLC purification. However, this step increases cost and reduces yield. Subsequent in vivo-purity processing subjects the duplexes to a series of dialysis and counterion exchange steps to remove toxic salts and solvents and lower the conductivity to physiological conditions. The resulting high-quality duplexes are ready for in vivo use regardless of whether HPLC purification is requested upstream of this process.
A. RNAi can be delivered using two different approaches: synthetic siRNA or siRNA expressed from plasmids or viral vectors (shRNA, miRNAi). siRNA-mediated gene targeting is generally the method of choice for the fast development of therapeutics. siRNA are easy to use, easy to design, and easy to synthesize. With RNAi vectors, the expression will be steadier as a result of the possibility of stable integration into the genome, and they have the ability to target nondividing cells such as stem cells, lymphocytes and neurons. The drawbacks are the danger of oncogenic transformation from insertional mutagenesis, and unanticipated toxicity from long-term silencing of human genes and/or having high amounts of siRNA inside the cell (Grimm D. et al.: Nature 441: 537-541 (2006) ).
A. Several different approaches have been used for siRNA delivery, including various local delivery techniques and systemic delivery.
A.Table 2 provides common nmole quantities in µg and mg.
Quantity | µmole | Ambion In Vivo siRNA | Stealth RNAi siRNA | ||
---|---|---|---|---|---|
µg | mg | µg | mg | ||
5 nmol | 0.005 | 68 | 0.068 | 79 | 0.079 |
100 nmol | 0.1 | 1354 | 1.354 | 1581 | 1.581 |
250 nmol | 0.25 | 3386 | 3.386 | 3953 | 3.953 |
1 µmol | 1 | 13,546 | 13.546 | 15,811 | 15.811 |
A. Please contact RNAiSupport@thermofisher.com regarding large scale in vivo RNAi.
When performing in vivo RNAi experiments with synthetic RNA duplexes, it is very important to have well-defined, non-toxic, and sterile starting material, compatible with physiological conditions. Thermo Fisher Scientific offers in vivo-ready siRNA reagents to maximize experimental success.
Our portfolio includes three types of siRNA processing and purification for in vivo RNAi research.
Ambion In Vivo siRNA molecules are chemically modified, 21-mer, double-stranded siRNAs that are recognized by the RNA-induced silencing complex (RISC) to mediate inhibition of a target gene. Proprietary chemical modifications allow Ambion In Vivo siRNAs to overcome many in vivo–specific obstacles, ensuring their effectiveness and stability in vivo. Ambion In Vivo siRNAs are at least 100x more stable in 90% mouse serum than unmodified siRNAs.
Ambion In Vivo siRNAs, the new standard for in vivo RNAi applications, offer:
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).
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.
Figure 2. Ambion In Vivo siRNA targeting FVII delivered with Invivofectamine 3.0 Reagent produce 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, a chemically modified 25-mer blunt-ended RNA duplex, has been chemically altered so only the antisense strand participates in the RNAi pathway, greatly decreasing the potential for off-target effects. Additionally, the chemical modification also allows Stealth RNAi siRNA to 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.
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. Following incubation samples were separated on a Novex 15% TBE-Urea polyacrylamide precast gel and stained with methylene blue.
To help ensure that the siRNA is delivered to targeted tissues, the following strategies have been used:
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 (human breast carcinoma) were transfected using Lipofectamine RNAiMAX. 24 hours post-transfection, fluorescent uptake was visualized and cells harvested for RNAi analysis.
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
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.
Learn more about lentiviral Pol II miR RNAi expression systems
The BLOCK-iT Pol II miR RNAi Expression Vectors offer significant advantages over shRNA vector technology. They retain the ability to achieve stable expression use of viral delivery, but also include capabilities for tissue-specific expression and multiple target knockdown from the same transcript.
The HiPerform Lentiviral Pol II miR RNAi Expression System with EmGFP(Figure 6) contains an mRNA stabilizing sequence (WPRE) and a nuclear import sequence (cPPT) which have generated up to 5-fold higher virus titers and EmGFP expression levels in many cell lines. Additionally, MultiSite technology allows you to express the EmGFP/miR RNAi cassette from CMV, EF-1a, or your own tissue-specific or other in vivo-appropriate promoter.
Figure 6. pLenti6/V5-DEST vector. The BLOCK-iT HiPerform Lentiviral POL II miR RNAi Expression System with EmGFP. The pLenti6.4/CMVor Ef-1a/V5-M5--GW/EmGFP-miR vector is driven by the CMV promotor, has the blasticidin resistance marker, and is available with co-cistronic EmGFP expression as a reporter.
Learn more about lentiviral Pol II miR RNAi expression systems
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 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 efficiencies. 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
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 |
For answers to additional questions, please refer to the Thermo Fisher Scientific technical support FAQ database or contact RNAiSupport@thermofisher.com to have a representative assist you.
A. Pre-designed Stealth RNAi and Ambion In Vivo siRNA can be ordered directly from our pre-designed siRNA search interface to target human, mouse, or rat genes.
Custom siRNA designs in vivo experiments can be ordered from two interfaced, depending on the product format being requested: Custom Ambion In Vivo siRNA is ordered from the GeneAssist Custom siRNA Builder, while Stealth RNAi is ordered using the BLOCK-iT RNAi Express for in vivo synthetics .
A. Chemically modified siRNA duplexes, such as Ambion In Vivo siRNA and Stealth RNAi, have a number of advantages over standard RNAi duplexes, including the minimization of off-target effects, enhanced stability, and reduced toxicity. For these reasons, chemically modified RNAi duplexes are recommended for in vivo RNAi experiments.
A. We have demonstrated that labeling Stealth RNAi duplexes does not hamper their knockdown potency. An alternative approach is to mix unlabeled duplexes with labeled control duplexes; this method is more commonly used with in vivo RNAi and allows progression to clinical research unhindered by questions about the possible effects of a dye.
A. Our mirVana Mimics and Inhibitors are available as pre-designed against mirBase v22 content in 250 nmol amounts, in vivo-ready and HPLC purified. Custom designs and other synthesis options can be ordered from our GeneAssist miRNA ordering tool.
A. Production of in vivo RNAi duplexes begins with standard synthesis of RNAi oligos using high-quality starting materials. The RNA oligos are then duplexed and desalted. At this point, the researcher can also request HPLC purification. However, this step increases cost and reduces yield. Subsequent in vivo-purity processing subjects the duplexes to a series of dialysis and counterion exchange steps to remove toxic salts and solvents and lower the conductivity to physiological conditions. The resulting high-quality duplexes are ready for in vivo use regardless of whether HPLC purification is requested upstream of this process.
A. RNAi can be delivered using two different approaches: synthetic siRNA or siRNA expressed from plasmids or viral vectors (shRNA, miRNAi). siRNA-mediated gene targeting is generally the method of choice for the fast development of therapeutics. siRNA are easy to use, easy to design, and easy to synthesize. With RNAi vectors, the expression will be steadier as a result of the possibility of stable integration into the genome, and they have the ability to target nondividing cells such as stem cells, lymphocytes and neurons. The drawbacks are the danger of oncogenic transformation from insertional mutagenesis, and unanticipated toxicity from long-term silencing of human genes and/or having high amounts of siRNA inside the cell (Grimm D. et al.: Nature 441: 537-541 (2006) ).
A. Several different approaches have been used for siRNA delivery, including various local delivery techniques and systemic delivery.
A.Table 2 provides common nmole quantities in µg and mg.
Quantity | µmole | Ambion In Vivo siRNA | Stealth RNAi siRNA | ||
---|---|---|---|---|---|
µg | mg | µg | mg | ||
5 nmol | 0.005 | 68 | 0.068 | 79 | 0.079 |
100 nmol | 0.1 | 1354 | 1.354 | 1581 | 1.581 |
250 nmol | 0.25 | 3386 | 3.386 | 3953 | 3.953 |
1 µmol | 1 | 13,546 | 13.546 | 15,811 | 15.811 |
A. Please contact RNAiSupport@thermofisher.com regarding large scale in vivo RNAi.
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
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.