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Order & InquiryTel: (713)732-2181 Fax: +1-866-747-4781E-mail: [email protected]
Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: [email protected]
RNA in vivo Transfection Reagent
Notice• To prepare endotoxin-free RNA, ONLY use distilled H2O to dissolve RNA.
• Prepare RNA/RNA in vivo Transfection Reagent mixture under sterile conditions.
• Ensure each amount/volume ratio of RNA/RNA in vivo
Transfection Reagent equals 2:1 (m/v).
• RNA and RNA in vivo Transfection Reagent require dilution
ONLY when used for intravenous (tail vein) injection.
DescriptionBiotool RNA in vivo Transfection Reagent is based on nanopolymer technology and is of the latest generation of non-viral transfection reagents.
This product is intended for research use only, not for use in diagnostic procedures!
Components
StorageThe product is shipped at room temperature. Store at 4-8°C for 12 months (For longer storage, keep at -20°C for 24 months). Avoid repeated freezing and thawing.
Regular recommended doses and volumes for administration are summarized in Table 1 based on injection route and animal used. Calculate amount/volume of each component according to RNA (1 µg /µL) : RNA in vivo Transfection Reagent (µL) : 5% glucose solution=2:1:5.
1. Suggested Injection Volume
Table1- Recommended Dose and Volume for Administration
Animal Administration Route Suggested RNAAmount
Adult Mouse
Nude Mouse
Adult Rat
Adult Rabbit
Maximum InjectionVolume
Tail Vein
Cerebral Ventricle
Peritoneum
Testes
Subcutaneous Tumor
Cerebral Ventricle
Tail Vein
Lung (intratracheal injection)
50 - 150 µg
1 - 2.5 µg
100 µg
3 - 5 µg
10 - 50 µg
2 - 5 µg
500 µg - 2.25 mg
300-700 µg
200 - 600 µL
5 µL
0.6 - 1mL
10 µL
100 µL
20 µL
1- 2 mL
300 - 700µL
RNA purity significantly affects transfection efficiency, endotoxin-free and high-purity RNA must be used. Prepare RNA in distilled water to a concentration of 1 µg/µL. Precipitation may occur if RNA is dissolved in non-distilled water (e.g. PBS).
Dilute RNA and RNA in vivo Transfection Reagent with the glucose solution, to ensure glucose final concentration is 5% after dilution. Table 2 contains a mock protocol for an RNA/RNA in vivo Transfection Reagent mixture for a mouse (weight= 20 g) by tail vein injection (injection volume=200 µl) based on a final dose of 2.5 mg/kg. The initial dose for tail-vein RNA delivery is 2.5 mg/kg. Recommended dose is 2.5-5 mg/kg. Generally, the higher the dose, the better the efficacy (when screened for no inflammatory response or lethality in animals).
2. Preparation of RNA
3. Tail vein injection
Generally, for 21 nt double-strand siRNA oligo, 1 OD=3.0 nmols=40 µg. But in some siRNA oligos, 1 OD=33 µg. Please refer to siRNA synthesis company materials before setting up injection protocol.
Note:
Use a concentration of RNA=1 µg/µL (RNA diluted by distilled water).For local injection, directly mix RNA/ RNA in vivo Transfection Reagent, then add glucose solution to the volume needed. No dilution is required for RNA or RNA in vivo Transfection Reagent before their blending.
Note:
Concentration of RNA=1 µg/µL (RNA diluted by distilled water).Note:
Table 2- Mock Protocol for RNA/ RNA in vivo Transfection Reagent mixture (200 µL)
Adult mouse
RNA in vivo Transfection
Reagent (diluted)
50 µL (50 µg) RNA
50 µL 10% (m/v) glucose solution
25 µL RNA in vivo Transfection Reagent
50 µL 10% (m/v) glucose solution
Distilled water 25 µL
Determine the maximum volume before injection. Calculate amount/ volume of each component according to RNA (1 µg/µL) : RNA in vivo Transfection Reagent (µL) : 5% glucose solution=2:1:5. Table 3 summarizes the protocol for making RNA/transfection reagent mixture by local injection (injection volume= 30 µL) as an example.
4. Local injection
Table 3- Protocol for RNA/ RNA in vivo Transfection Reagent mixture (30 µl)
7.5 µL (7.5 µg)
18.75 µL
3.75 µL
RNA
5% Glucose solution
RNA in vivo Transfection Reagent
Component
RNA in vivo transfection
reagent
20 injections(0.5 ml)
100 injections (2.5 ml)
1000 injections(25 ml)
Cat#: B45212 Cat#: B45215 Cat#: B45218
This product contains no biologically hazardous or chemically toxic compounds. Rinse with clean water if splashed.
Order & InquiryTel: (713)732-2181 Fax: +1-866-747-4781E-mail: [email protected]
Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: [email protected]
The following protocol is given for intravenous injection (tail vein) for a mouse weighing 20 g in a final volume of 200 µL. Please adjust YOUR protocol accordingly based on route, dose, volume and animal used for injection.
Dilute RNA with endotoxin-free H2O to a concentration of 1 µg/µL. Add 50 µL RNA solution into 50 µL 10% glucose solution to obtain a 100 µL solution with a final concentration of 5% glucose. Vortex gently and centrifuge briefly.
Generally, robust gene delivery or silencing can be detected 12-48 h post injection, depending on the method of injection and the target organ.
Dilute 25 µL of RNA in vivo Transfection Reagent in 50 µL of 10% glucose. Add 25 µL H2O to obtain a 100 µL solution with a final concentration of 5% glucose. Vortex gently and spin down briefly.
Incubate the mixture at Room Temperature for 15 min.
Immediately transfer diluted RNA in vivo Transfection Reagent (100 µL) into diluted RNA (100 µL). Mix well by thoroughly vortexing then spin down briefly.
Prepare fresh RNA/transfection reagent mixture before each use.The mixture is not recommended for long storage periods and is stable for up to 24 h at 4℃.
Note:
Protocol
1. RNA dilution
6. Gene Expression Detection
7. Long Period Administration
2. RNA in vivo Transfection Reagent dilution
3. Mixture
4. Incubation
5. Animal Injection
Inject at the dorsal surface of the 1/3 part of distal end of the tail. Stop injection if resistance or a slight bump exists. Inject at a new site and DO NOT push the syringe plunger with brute force. This could cause the transfection mixture to be detained in tail, which may result in fester.Press pinhole for over 10 seconds for effusion of transfection mixture.Scale up the administration dose to obtain higher transfection efficiency if the animal presents satisfying tolerance.For local injections, scale up final injection volume if possible, to increase transfection efficiency.
a.
b.
c.
d.
e.
Generally, the best time of sampling for one time injection is 12-48h after the injection. Multiple injections are recommended for maintenance of long period efficacy. In most cases, about one injection per 3-4 days is suggested but the injection frequency could be prolonged to one injection per 7days for different experiments.
Troubleshooting
Possible Reason Suggested Improvement Problem
RNA not dissolved in distilled H2O
(e.g. Tris-EDTA)Prepare fresh culture.
Precipitate formation in transfection
mixture
Low transfection efficiency
Inflammation response or
lethality of animal
Excessive RNA concentration in
transfection mixture
Transfection mixture froze or was stored at low
temperature for a long period of time
Low administration dose
Injection manipulation error
Ensure RNA concentration in transfection mixture ≤ 0.5 µg/µL
before injection
Freshly prepare transfection mixture
and use immediately
Scale up administration dose in the case
of exclusion of animal lethality
Inject the transfection mixture at correct
site by correct method
Excessive RNA amountScale down RNA amount and
adjust the volume of transfection reagent accordingly
Endotoxin in transfection mixture
Prepare endotoxin-free RNA
Pass transfection mixture through a 0.22 µm
sterile filter before injection
1. Dilute 50μL(1μg/μL) siRNA or miRNA in 50μL 10% glucose.
Vortex gently
2. Dilute 25μL RNA in vivo Transfection Reagent in 50μL 10% glucose. Add 25uL H2O.
3. Add diluted RNA in vivo Transfection Reagent to diluted RNA.
4. Vortex gently and incubate 15min at room temperature.
5. Inject using the appropriate route.
Delivery into the lumbar
Delivery into the liver,kidney,spleen,pancreas
Delivery into the retro-orbital
Delivery into the cerebralNasal instillation
Intraperitoneal injectionDelivery into the bladder
Direct intratumoral injection
Tail vein injection