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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: order@biotool.com

Order & InquiryTel: +49-89-46148500 Fax: +49-89-461485022E-mail: eu.order@biotool.com

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