rna-invivotransfer® in vivo transfection reagent for rna...

User Manual

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RNA-Invivotransfer® in vivo transfection reagent for RNA

Description ............................................................................................................ 2

Application ............................................................................................................ 2

Important Guidelines .............................................................................................2

Protocol ................................................................................................................ 4

Trouble Shooting .................................................................................................. 6

Storage and Safety ………………………………….……………………………….... 7

Quality Certification ………………………………….………………………………....7

Contents1. Description

2. Application

3. Important Guidelines

Biotool Co.,Ltd, a brand of Selleckchem LCC, manufactures professional transfection reagents. Biotool RNA-Invivotransfer® in vivo transfection reagent for RNA is based on nanopolymer technology and is of the latest generation of non-viral transfection reagents. RNA-Invivotransfer® is a patent-pending, animal-origin-free formulation providing the following advantages:

Effective and reproducible in vivo RNA delivery with low toxicity and high efficiency.Non-viral vector for gene transfer (safe for users).The simplest and safest approach to deliver genetic material in vivo.Raises no detectable inflammatory response in host animals.

The product is designed for RNA in vivo transfection,for research interests such as:

Caution: This product is intended for research use only, not for use in diagnostic procedures!

1. Suggested RNA AmountThe 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). Regular recommended doses and volumes for administration are summarized in Table 1 based on injection route and animal used.

Table1- Recommended Dose and Volume for Administration

1. in vivo study of gene therapy for animals.2. in vivo study of RNA interference for animals.3. in vivo study of protein functions for animals.

1.

2.3.4.

1 RNA-Invivotransfer® in vivo transfection reagent for RNA RNA-Invivotransfer® in vivo transfection reagent for RNA 2

To prepare endotoxin-free RNA, ONLY use distilled H2O to dissolve RNA.

Prepare RNA/RNA-Invivotransfer® mixture under sterile conditions.Ensure each amount/volume ratio of RNA/ RNA-Invivotransfer® equals 2:1 (m/v).RNA and RNA-Invivotransfer® require dilution ONLY when used for intravenous (tail vein) injection .Adjust YOUR protocol based on the method, dose, final volume and animal used before each injection.

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Animal Administration Route Suggested RNA Amount

Adult Mouse

Nude Mouse

Adult Rat

Adult Rabbit

Maximum Injection Volume

Tail VeinCerebral Ventricle

PeritoneumTestes

Subcutaneous TumorCerebral Ventricle

Tail VeinLung (intratracheal injection)

50 - 150 µg1 - 2.5 µg

100 µg3 - 5 µg

10 - 50 µg2 - 5 µg

500 µg - 2.25 mg300-700 µg

200 - 600 µL5 µL

0.6 - 1mL10 µL100 µL20 µL

1- 2 mL300 - 700µL

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4. ProtocolNote: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:Concentration of RNA=1µg/µL (RNA diluted by distilled water).

Note:Use a concentration of RNA=1 µg/µL (RNA diluted by distilled water).For local injection, directly mix RNA/ RNA-Invivotransfer®, then add glucose solution to the volume needed. No dilution is required for RNA or RNA-Invivotransfer® before their blending.

2. Preparation of RNARNA purity significantly affects transfection efficiency, endotoxin-free and high-purity RNA must be used. Prepare RNA in distilled water to a concentration of 1 – 4 µg/µL. Precipitation may occur if RNA is dissolved in non-distilled water (e.g. PBS).

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.

1. RNA dilutionDilute 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.

2. RNA-Invivotransfer® dilutionDilute 25 µL of RNA-Invivotransfer® 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.

3. MixtureImmediately transfer diluted RNA-Invivotransfer® (100 µL) into diluted RNA (100 µL). Mix well by thoroughly vortexing then spin down briefly.

4. IncubationIncubate the mixture at Room Temperature for 15 min.

Notes：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℃.

5. Animal Injection

3. Suggested RNA-Invivotransfer AmountGenerally, RNA (µg) : RNA-Invivotransfer® (µL)=2:1 (e.g.: 100 µg: 50 µL).

4. Tail vein injectionDilute RNA and RNA-Invivotransfer® with the glucose solution, to ensure glucose final concentration is 5% after dilution. Table 2 contains a mock protocol for an RNA/ RNA-Invivotransfer® mixture for a mouse (weight= 20 g) by tail vein injection (injection volume= 200µl) based on a final dose of 2.5mg/kg.


Table 2- Mock Protocol for RNA/ RNA-Invivotransfer® mixture (200µl)

Adult mouse

RNA-Invivotransfer® (diluted)

50 µL (50 µg) RNA50 µL 10% (m/v) glucose solution25 µL RNA-Invivotransfer50 µL 10% (m/v) glucose solutionDistilled water 25µL

5. Local injectionDetermine the maximum volume before injection. Calculate amount/volume of each component according to RNA (µg) : RNA-Invivotransfer® (µL)=2:1. Table 3 summarizes the protocol for making RNA/transfection reagent mixture by local injection (injection volume= 30 µL) as an example.

Table 3- Protocol for RNA/ RNA-Invivotransfer® mixture (30µl)

15 µL (15µg)7.5 µL7.5 µL

RNAGlucose solutionRNA-Invivotransfer

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Prepare the RNA/ RNA-Invivotransfer® mixture under sterile conditions.Prior to injecting the mixture, ensure that the mixture is at room temperature.For intravenous injection (tail vein), dilute RNA and RNA-Invivotransfer®

accordingly, before making a mixture.For local injection, no dilution is required for RNA or RNA-Invivotransfer®. Directly mix RNA and RNA-Invivotransfer, then add glucose solution to the volume needed.

5. Trouble-shooting

6. Storage and SafetyAttention for intravenous injection:

6. Gene Expression DetectionGenerally, robust gene delivery or silencing can be detected 12-48 h post injection, depending on the method of injection and the target organ.

7. Long Period AdministrationGenerally, the efficacy of each administration lasts for 1 to 2 weeks. Multiple injections are recommended for maintenance of long period efficacy. In most cases, one inject per week is suggested but the injection frequency shall be shortened or prolonged according to specific experimental purposes.

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.

The 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.

This product contains no biologically hazardous or chemically toxic compounds. Rinse with clean water if splashed.

7. Quality CertificationBiotoolTM implements quality inspection and transfection performance assay for every batch of RNA-Invivotransfer®.


a.b.c.

d.e.

f.

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

rna-invivotransfer® in vivo transfection reagent for rna...

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