in vivo RNAi Protocols

Refer to the following supplemental protocols for special consideration for in vivo experiments. For questions about a protocol, please contact our Technical Support to have a representative guide you through the procedure.

• Resuspension of Stealth RNAi siRNA for in vivo applications
• Measuring RNA concentration
• Injection techniques
• Blood collection
• Tissue harvest - RNA extraction
• Tissue sectioning
• Protein extraction from tissues
• Tissue preparation for flow cytometry analysis

In vivo Purity Stealth RNAi siRNA and BLOCK-iT 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.

Table 1. Desalted purity: recommended resuspension volume for 5mg/mL final concentration.

Table 2. HPLC purity: recommended resuspension volume for 5mg/mL final concentration.

NOTE: The recommended resuspension volumes are for an RNAi molecule with 50 % GC. The molecular weight of RNAi molecules varies slightly depending on the GC content, but these differences are negligible for in vivo RNAi experiments.

If desired, measure RNA concentration using UV absorbance at 260 nm (A₂₆₀). Dilute the RNA solution in resuspension buffer or water, 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

NOTE: The formulas for Stealth RNAi siRNA and Block-iT siRNA are slightly different due to chemical and size differences.

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.

Mouse injection points

IP - Intraperitoneal

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

IV - Intravenous

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 a better visualization of the vein and dilation, warm up the vein to ~37º 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 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 previous site. Upon completion remove needle and apply pressure to injection site.

IN- Intranasal

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 ul 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.

IT-Intratumoral

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 ´ 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 of 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 higher amount of blood is needed or multiple draws need to be performed.

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 x 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 assay procedure provided with kit for appropriate dilutions.

Plasma

Remove the cells from the samples by centrifugation at 2,000 x 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 assay procedure provided with kit for appropriate dilutions.

Blood analysis

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

References

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.

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 degrees. A tissue homogenizer or rotor-stator can also be used.
2. For harder tissues (tumors, lungs), perform 3 cycles of 60 seconds each at 6m/s. For softer tissues (brain, liver), 1 cycle of 60sec at 4.5m/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 available there.
3. Determine the quality and quantity of the purified RNA using UV absorbance at 260 nm or Quant-iT RNA Assay Kit (cat. no.Q33140) 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 GreenER qPCR Super Mix.

Slide Preparation

If you are preparing your own slides, pre-coat slides with HistoGrip (Invitrogen cat no. 008050 ) 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

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

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). Store frozen tissue blocks at -70ºC until you are ready for tissue sectioning. 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. Dry tissue sections at room temperature for 30 minutes. If desired, store slides at -70ºC before fixing. If slides are stored at -70ºC, warm the slides to room temperature before the fixing step. Place the slides in 100% acetone at 4ºC for 10 minutes to fix the sections. Remove slides from acetone and air dry for 10-30 minutes. Circle each tissue section using the Mini PAP Pen. Store at -70ºC 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. Obtain or prepare the formalin-fixed paraffin embedded sections of choice. Dry slides containing 4 µm formalin-fixed paraffin embedded sections in a 55ºC oven for 2 hours or overnight (do not allow the temperature to exceed 60ºC). Store the slides containing the formalin-fixed paraffin embedded tissue sections at room temperature until needed. Place slides in xylene for 5 minutes at room temperature. Remove slides and place in xylene a second time for an additional 5 minutes. Remove slides and place in 100% ethanol 2 times for 5 minutes each time. Remove slides and place in 95% ethanol for 5 minutes.  Remove slides and place in 80% ethanol for 5 minutes. Remove slides and place in PBS for 10 minutes.  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.

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. Typically, cut the tissue into small pieces and place 100 mg tissue in a microcentrifuge tube. Add 1 ml Cell Extraction Buffer containing Protease Inhibitor Cocktail. Homogenize the tissue using a pestle that fits into the microcentrifuge tube. Incubate the samples on ice for 10 minutes with intermittent vortexing. Centrifuge the lysate at 10,000 x g for 5-10 minutes to remove any particulate material. Transfer and aliquot the supernatant to sterile microcentrifuge tubes and proceed to Preparing Samples for SDS-PAGE after protein estimation, or store aliquots at -80ºC. For more detailed information follow protocol described in the kit (cat. No. WFGE01).

Euthanize the mouse by an atraumatic method such as anesthetic overdose by CO₂ narcosis. Collect the tissue of interest and place it in cold PBS. Mince the tissue into ~1mm3 cubes using a razor blade in a 35mm Petri dish containing 1-2ml of digesting media (17703-034) . Incubate 15 to 45 minutes depending of the hardness of the tissue and pipettes up and down every 15 minutes using a 5ml pipettors. After incubation, add 4-5ml of PBS and strain (100um) the slurry, using minimum force until they will pass through the strainer, into a 10ml conical tube. Wash the cells 2 to 3 times with PBS, by spinning the cells at 1,000g for 5 minutes and discarding  the supernatant between washes. If the pellet is significantly red, wash the pellet with erythrocytes lysis buffer (L5) from the PureLink Total RNA Blood Kit (K1560-01). To the pellet of cells, add 500ul of (L5). Incubate for 10 minutes on ice. Vortex the tube briefly 2-3 times during the incubation step to allow complete lysis of erythrocytes. The solution turns translucent. Centrifuge the tube at 4°C at 400 x g for 10 minutes. Remove the supernatant completely and discard the supernatant.  Resuspend the cell pellet in PBS and proceed with the wash. The cell pellet should be white with no traces of red.  Resuspend the pellet into 0.5% PFA and strained (50um) 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 the extent Invitrogen provides general guidelines for using siRNA in animals, the company makes no guarantees concerning use of these products or guidelines in animal studies.