model animal in laboratory setting

Solutions for successful in vivo RNAi

We offer solutions that overcome many of the inherent challenges of in vivo RNAi work by providing:

  • 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
  • Pre-designed siRNA for human, mouse, and rat, or custom-designed to meet your needs

In vivo RNAi experiments are more challenging than their in vitro 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.

Search for pre-designed siRNA

Which In Vivo siRNA is right for you?

  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

Order RNAi controls

Considerations for in vivo siRNA experiments

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.

  • 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 <200µS and sterile filtration.

Ambion In Vivo siRNAs

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:

  • High stability against nucleases
  • No induction of the interferon response
  • Easy tracking of administered siRNAs
  • Combined knockdown effectiveness and stability
     

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.

Less siRNA required to achieve effective knockdown

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.

Stealth RNAi siRNA

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.

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-QDots (Figures 4, 5). This can be ordered using our custom in vivo Stealth RNAi ordering tool .

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.

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.

Learn more about lentiviral Pol II miR RNAi expression systems

BLOCK-iT Pol II miR RNAi Expression Vectors

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.

  • Achieve up to 5x higher titers (measured by GFP) allowing more cells to be transduced or higher multiplicities of infection (MOIs) to be employed
  • Incorporate your own tissue-specific or other in vivo appropriate promoter
  • Track expression through co-cistronic expression with EmGFP (Figure 7)
  • Knockdown more than one gene simultaneously through expression of multiple miRNAs from a single transcript

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 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

  • 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            

In vivo RNAi Frequently Asked Questions

Looking for an answer to your question? Browse the topics listed.

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.

Q. How can I order in vivo siRNA?

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 .

Q. Should I use chemically modified duplexes for my in vivo RNAi experiments?

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.

Q. Should I use labeled or unlabeled duplexes for my 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.

Q. I would like to modulate the expression of microRNAs in vivo. Do you have suitable mimics and inhibitors for this?

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.

Q. I am planning on using siRNA for my in vivo experiments, what purity should they be?

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.

Q. Should I use siRNA or vectors for in vivo RNAi?

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) ).

Q. How should I deliver my in vivo RNAi molecules?

A. Several different approaches have been used for siRNA delivery, including various local delivery techniques and systemic delivery.

Q. What are the µg or mg amounts that correspond to the nmole quantities offered for in vivo synthetic siRNA?

A. Table 2 provides common nmole quantities in µg and mg.

Table 2. nmol 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

Q. I want to order larger scales than the standard offering, whom do I contact?

A. Please contact RNAiSupport@thermofisher.com regarding large scale in vivo RNAi.

Considerations for in vivo siRNA experiments

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.

  • 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 <200µS and sterile filtration.

Ambion In Vivo siRNAs

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:

  • High stability against nucleases
  • No induction of the interferon response
  • Easy tracking of administered siRNAs
  • Combined knockdown effectiveness and stability
     

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.

Less siRNA required to achieve effective knockdown

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.

Stealth RNAi siRNA

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.

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-QDots (Figures 4, 5). This can be ordered using our custom in vivo Stealth RNAi ordering tool .

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.

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.

Learn more about lentiviral Pol II miR RNAi expression systems

BLOCK-iT Pol II miR RNAi Expression Vectors

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.

  • Achieve up to 5x higher titers (measured by GFP) allowing more cells to be transduced or higher multiplicities of infection (MOIs) to be employed
  • Incorporate your own tissue-specific or other in vivo appropriate promoter
  • Track expression through co-cistronic expression with EmGFP (Figure 7)
  • Knockdown more than one gene simultaneously through expression of multiple miRNAs from a single transcript

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 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

  • 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            

In vivo RNAi Frequently Asked Questions

Looking for an answer to your question? Browse the topics listed.

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.

Q. How can I order in vivo siRNA?

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 .

Q. Should I use chemically modified duplexes for my in vivo RNAi experiments?

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.

Q. Should I use labeled or unlabeled duplexes for my 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.

Q. I would like to modulate the expression of microRNAs in vivo. Do you have suitable mimics and inhibitors for this?

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.

Q. I am planning on using siRNA for my in vivo experiments, what purity should they be?

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.

Q. Should I use siRNA or vectors for in vivo RNAi?

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) ).

Q. How should I deliver my in vivo RNAi molecules?

A. Several different approaches have been used for siRNA delivery, including various local delivery techniques and systemic delivery.

Q. What are the µg or mg amounts that correspond to the nmole quantities offered for in vivo synthetic siRNA?

A. Table 2 provides common nmole quantities in µg and mg.

Table 2. nmol 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

Q. I want to order larger scales than the standard offering, whom do I contact?

A. Please contact RNAiSupport@thermofisher.com regarding large scale in vivo RNAi.

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If you have any questions on products or ordering, please contact us at RNAiSupport@thermofisher.com