In Vivo RNAi Protocols

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules. This natural mechanism is harnessed in research and therapeutic applications to silence specific genes, offering a powerful tool for studying gene function and developing treatments for genetic disorders. The following RNAi protocols help guide researchers in effectively implementing RNAi techniques, including the design and delivery of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) into cells. These protocols cover various aspects, such as selecting target sequences, optimizing transfection or transduction methods, and analyzing gene knockdown efficiency.

Our available in vivo RNAi protocols extend these techniques to living organisms, helping enable gene silencing in specific tissues or cell types within the context of a whole organism. These research methods are important for studying gene function in physiological conditions, verifying specific targets, and researching the development of RNAi-based therapies. These in vivo RNAi protocols involve additional considerations, such as delivery methods (e.g., viral vectors, nanoparticles), dosage, and suggestions for minimizing off-target effects, to help ensure effective and specific gene silencing in vivo.

For questions about a protocol, please contact our Technical Support.

In vivo ready siRNA duplexes are specifically formulated for use in animals.

Resuspend the RNA duplex in UltraPure DNase/RNase-free distilled water or appropriate DNase/RNase-free buffer (e.g., PBS, Ringer’s solution, 0.9% NaCl). A 5 mg/mL stock solution is recommended for in vivo RNAi experiments. Table 1 and Table 2 specify the recommended resuspension volume for in vivo Purity Stealth RNAi siRNA and BLOCK-iT siRNA.

Measure RNA concentration using UV absorbance at 260 nm (A₂₆₀). Dilute the RNA solution in resuspension buffer or water, and mix well. Measure the A₂₆₀ of the dilution in a spectrophotometer blanked against dilution buffer (using a cuvette with a 1 cm optical path length). Calculate RNA concentration using the appropriate formula:

Stealth RNAi siRNA
RNA concentration (μg/mL) = A₂₆₀ (OD₂₆₀ units) x 44 ((μg/mL)/OD unit) x dilution factor

BLOCK-iT RNAi
RNA concentration (μg/mL) = A₂₆₀ (OD₂₆₀ units) x 41 ((μg/mL)/OD unit) x dilution factor

Animals should be handled and experiments conducted according to national regulations and approved by the local experiments ethical committee. All persons handling animals should be properly trained by the local facility. Weigh the animals prior to injection and maintain a body-weight record over the course of the experiment.

Intraperitoneal (IP)

Restrain the mouse and expose the abdomen. Disinfect the injection site by swabbing the area with an alcohol swab, insert the needle into the abdomen at a 45-degree angle, and inject slowly (20 μL/sec).

Intravenous (IV)

The mouse should be restrained using a mouse restraining device or by injection of an anesthetic in the lower flank of the mouse. Alternatively, a tail veiner (Braintree Scientific) can be used to better visualize the tail vein and optimize the success of the injection. Disinfect the site of injection and slightly rotate the tail to visualize the vein. For better visualization of the vein and dilation, warm up the vein to ~37°C using either a water bath or a heat lamp. Once the vein has been located, disinfect the site of injection and insert the needle at a slight angle. Inject slowly (~20 μL/sec) and watch for clearing of the blood. If the resistance increases, and a slight bulge appears in the tail, remove the needle and repeat the process proximal to the previous site. Upon completion, remove the needle and apply pressure to the injection site.

Intranasal (IN)

For this injection, mice should be anesthetized by injecting 0.2–0.3 mL of anesthetic in the lower flank of the mouse. Place the mouse on its back over a warm pad or under a warming lamp. Inject 20 μL in each nostril using a 20 μL pipettor. Inject slowly and wait about 1 minute between injections to help recovery if you observe shortening of breath.

Intratumoral (IT)

The mouse should be restrained using a mouse restraining device or by injection of an anesthetic in the lower flank of the mouse. When the subcutaneous tumor areas reach approximately 3 X 3 mm² in size, the mice are ready for injection. The volume of injection is typically 0.5 μL/mm³. Use forceps to hold the tumor. Disinfect the site of injection and insert the needle directly into the tumor and penetrate as deeply as possible without passing through the tumor. Slowly push the solution into the tumor. Slowly pull the needle back, but not out of the tumor, change the direction of the needle inside the tumor and then push in. After injection, leave the needle in the tumor for about 20 seconds, and then slowly pull out and pinch the opening with fine forceps to avoid leakage.

The method for blood collection depends on the amount of blood is required per sample. If a few drops are needed, tail clipping will work. However, retro-orbital or saphenous vein draw should be used if a higher amount of blood is needed or if multiple draws need to be performed [1].

Serum

Samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to sit at room temperature for 15–30 minutes to clot. Spin at 1,000–2,000 × g for 10 minutes in a 4°C refrigerated centrifuge to separate the cells. Transfer the supernatant to a clean, chilled polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2–8°C while handling. If serum is to be analyzed at a later date, apportion the serum into 0.5 mL aliquots and store at –80°C. Avoid multiple freeze-thaw cycles. When possible, avoid the use of hemolyzed or lipemic sera. Upon thawing, it is recommended that the samples be clarified by centrifugation (14,000 rpm for 10 minutes) and/or filtered prior to analysis to prevent clogging of the filter plates and/or probe. Follow the assay procedure provided with the kit for appropriate dilutions.

Plasma

Remove the cells from the samples by centrifugation at 2,000 × g for 10 minutes in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete platelets from the sample. Transfer the supernatant to a clean, chilled polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2–8°C while handling. If the plasma is to be analyzed at a later date, apportion into aliquots in polypropylene microcentrifuge tubes and store at –80°C. Avoid multiple freeze-thaw cycles. When ready to analyze, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation at 14,000 rpm for 10 minutes at 4°C in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.

Blood analysis

Extract RNA from blood and use qRT-PCR for siRNA biodistribution study and Luminex assay to measure IFN response.

1. Homogenize 50–100 mg of tissue in 1 mL TRIzol Reagent using lysing matrix D on the FastPrep-24 Instrument (MP Biomedical) at 4°C. A tissue homogenizer or rotor-stator can also be used.
2. For harder tissues (tumors, lungs), perform 3 cycles of 60 seconds each at 6 m/s. For softer tissues (brain, liver), 1 cycle of 60 s at 4.5 m/s is sufficient to completely dissociate the tissue. Add 0.2 mL of chloroform directly into the tube and process following the protocol described in the PureLink Micro-to-Midi RNA Purification System manual.
3. Determine the quality and quantity of the purified RNA using UV absorbance at 260 nm or Quant-iT RNA Assay Kit or run on agarose E-gels.
4. After quantification, use 750 ng of total RNA for first strand synthesis using Superscript III RT kit and qPCR analysis performed using SYBR Green qPCR Super Mix.

Tissue sectioning is an important technique in histology and pathology that involves slicing thin sections of biological tissues for microscopic examination. This process is essential for visualizing the cellular and molecular effects of experimental treatments, such as those referenced here. In the context of in vivo RNAi, tissue sectioning allows researchers to assess the distribution and efficacy of RNAi-mediated gene silencing within specific tissues and cell types. By examining tissue sections, scientists can observe the morphological changes, gene expression patterns, and potential off-target effects resulting from RNAi treatments.

Slide preparation

If you are preparing your own slides, pre-coat slides with HistoGrip or 0.1% poly-L-lysine in water, then air dry. Commercially available pre-coated glass slides are available and can be used to mount frozen or formalin-fixed paraffin embedded tissue sections.

Frozen tissue

An example protocol for preparing frozen tissue samples is described below, but this is only an example. If you have optimized protocols in the laboratory for your sample type, use the optimized protocol.

1. Snap freeze fresh tissues in cryomolds containing OCT (Optimal Cutting Temperature) compound (a solution of glycols and resins which provides an inert matrix for sectioning).
2. Store frozen tissue blocks at –70°C until you are ready for tissue sectioning.
3. For sectioning, allow the frozen tissue block to equilibrate to the cryostat temperature, cut 4–20 μm cryostat sections, and mount on coated glass slides.
4. Dry tissue sections at room temperature for 30 minutes. If desired, store slides at –70°C before fixing. If slides are stored at –70°C, warm the slides to room temperature before the fixing step.
5. Place the slides in 100% acetone at 4°C for 10 minutes to fix the sections.
6. Remove slides from acetone and air dry for 10–30 minutes. Circle each tissue section using the Mini PAP Pen.
7. Store at –70°C until use or wash the slide in PBS for 10 minutes and with staining or mounting.

Paraffin embedded sections: Deparaffinization and rehydration

To use the formalin-fixed paraffin embedded sections for immunohistochemical staining, deparaffinization with xylene needs to be performed followed by rehydration in a graded series of alcohol as described in the example protocol below.

1. Obtain or prepare the formalin-fixed paraffin embedded sections of choice.
2. Dry slides containing 4 μm formalin-fixed paraffin embedded sections in a 55°C oven for 2 hours or overnight (do not allow the temperature to exceed 60°C). Store the slides containing the formalin-fixed paraffin embedded tissue sections at room temperature until needed.
3. Place slides in xylene for 5 minutes at room temperature.
4. Remove slides and place in xylene a second time for an additional 5 minutes.
5. Remove slides and place in 100% ethanol 2 times for 5 minutes each time.
6. Remove slides and place in 95% ethanol for 5 minutes.
7. Remove slides and place in 80% ethanol for 5 minutes.
8. Remove slides and place in PBS for 10 minutes.
9. Drain any excess reagent by tapping the edge of the slide on paper towels and wipe the area near the tissue sections with a laboratory wipe. Circle each tissue section using the Mini PAP Pen.

Protein extraction from tissues is a technique in molecular biology that involves isolating proteins from biological samples for downstream analysis. This process is relevant in the context of in vivo RNAi protocols, where researchers aim to understand the impact of gene silencing on protein expression and function within specific tissues. After administering RNAi treatments to an organism, protein extraction allows scientists to quantify and analyze the levels of target proteins to determine the efficacy of gene knockdown. By comparing protein expression in treated versus control tissues, researchers can verify the success of RNAi-mediated silencing at the protein level, which is essential for confirming the biological effects of gene knockdown. Additionally, protein extraction enables the study of downstream effects and pathways affected by the silenced gene, offering insights into the molecular mechanisms underlying observed phenotypes. This technique, combined with methods such as western blotting and mass spectrometry, offers a comprehensive approach to evaluating the outcomes of in vivo RNAi experiments.

Sample preparation

Proper sample preparation is key to the success of a western blot analysis experiment. Various factors affect the design of a sample preparation protocol. Due to the large variety of proteins present in different cells and tissues, it is not possible to have a single sample preparation protocol that is suitable for all proteins. Based on the starting material and goal of the experiment, the sample preparation protocol needs to be determined empirically. The sample preparation conditions may also be optimized based on your initial results. If an optimized sample preparation protocol exists in the laboratory for your specific samples, use the optimized protocol. General guidelines are provided below to prepare samples from various sources, and example procedures are provided.

Mammalian tissue samples

A protocol for preparing mammalian tissue lysate from 100 mg tissue using 1 mL Cell Extraction Buffer is described below. This protocol is suitable for use with a variety of tissue types; some optimization may be required for some tissues.

1. Cut the tissue into small pieces and place 100 mg tissue in a microcentrifuge tube.
2. Add 1 mL Cell Extraction Buffer containing Protease Inhibitor Cocktail.
3. Homogenize the tissue using a pestle that fits into the microcentrifuge tube.
4. Incubate the samples on ice for 10 minutes with intermittent vortexing.
5. Centrifuge the lysate at 10,000 × g for 5–10 minutes to remove any particulate material.
6. Transfer and aliquot the supernatant to sterile microcentrifuge tubes and proceed to preparing samples for SDS-PAGE after protein estimation, or store aliquots at -80°C.

1. Euthanize the mouse by an atraumatic method such as anesthetic overdose by CO₂ narcosis.
2. Collect the tissue of interest and place it in cold PBS. Mince the tissue into ~1 mm³ cubes using a razor blade in a 35 mm Petri dish containing 1–2 mL of digesting media.
3. Incubate for 15 to 45 minutes depending on the hardness of the tissue and pipette up and down every 15 minutes using a 5 mL pipettor.
4. After incubation, add 4–5 mL of PBS and strain (100 µm) the slurry, using minimum force until it passes through the strainer, into a 10 mL conical tube.
5. Wash the cells 2 to 3 times with PBS, by spinning the cells at 1,000 g for 5 minutes and discarding the supernatant between washes. If the pellet is significantly red, wash the pellet with erythrocyte lysis buffer (L5) from the PureLink Total RNA Blood Kit.
6. To the pellet of cells, add 500 µL of L5. Incubate for 10 minutes on ice.
7. Vortex the tube briefly 2–3 times during the incubation step to allow complete lysis of erythrocytes. The solution should turn translucent.
8. Centrifuge the tube at 4°C at 400 x g for 10 minutes. Remove the supernatant completely and discard the supernatant.
9. Resuspend the cell pellet in PBS and proceed with the wash. The cell pellet should be white with no traces of red.
10. Resuspend the pellet in 0.5% PFA and strain (50 µm) before flow cytometry analysis.

Special note and disclaimer

Literature describing in vivo delivery of siRNA and modified siRNA has become more abundant. However, the applications and methods described often vary. To this extent, we provide general guidelines for using siRNA in vivo, we make no guarantees concerning use of these products or guidelines in animal studies.

• RNAi: A Four-Step Workflow
• RNAi How-To for New Users

1. Van Herck H, Baumans V, Brandt CJWM, Boere HAG, Hesp APM, Van Lith HA, Schurink M, Beynen AC (2001). Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables. Laboratory Animals 35: 131–139.