The Transfection &Gene Expression Experts

Avalanche®-in vivo Transfection Reagent
Cat. No. EZT-VIVO-1

Size: 200 µl
600 µlStore at 4°C

DESCRIPTION
Avalanche®-in vivo Transfection Reagent (Hereafter “Avalanche®-in vivo”) is an extremely
powerful reagent for in vivo gene functional studies and RNA interference. The proprietary
mixture of lipids and polymers is able to form compact complexes with nucleic acids, andmove the nucleic acids into cells via endocytosis. In addition to efficiently moving nucleicacids into the cells, Avalanche®-in vivo is also being able to protect the nucleic acids fromlysosomal degradation, which ensures the efficient release of the nucleic acids fromendosomes and maximizes the function of those nucleic acids. Avalanche®-in vivo has been
proved to be able to perform the maximum delivery of DNA, shRNA, siRNA, oroligonucleotides into different organs in the body. No significant pro-inflammatory
responses have been detected using Avalanche®-in vivo. Avalanche®-in vivo can beadministered via various routes, such as intravenous, intraperitoneal, and intratumoral
injection etc. Avalanche®-in vivo Transfection Reagent is the best choice for delivering
nucleic acids in vivo.
Features: Efficient delivery of DNA, shRNA, siRNA or oligonucleotides in vivo
 200 µl reagent for 20 transfections in mouse Efficient delivery to the lung, liver, kidney, and spleen, liver, pancreas and certain
types of tumors via systemic administration
 Delivery via multiple modes of administration in many species Avalanche-in vivo®/nucleic acid conjugated complexes are stable in serum for 16h
 No detectable inflammatory responses
 Efficient siRNA and plasmid DNA delivery via direct subcutaneous tumor injection(multiple tumor types)
 Reproducible results
 Applicable for plasmid DNA/siRNA co-injection
 Developed and manufactured by EZ Biosystems LLC

Avalanche®-in vivo Transfection Reagent

IN VIVO DELIVERY PROTOCOL
1.1 Before You Start:
 Nucleic acids should be prepared in low salt buffer, since high salt content in thenucleic acid preparation may lead to precipitation upon complexes formation. For
DNA delivery, the best results are achieved with high quality DNA in endotoxin freeddH2O. For siRNA, order high quality grade siRNA (PAGE or HPLC purification).
 To avoid precipitation, the nucleic acid preparation should be at high concentration (ifpossible for DNA 3-7 μg/μl and for siRNA 5-10 μg/μl).
 The concentration of nucleic acids in the final injection solution should not exceed 0.5μg/μl.
 The preparation of Avalanche®-in vivo /nucleic acid complexes should be performed ina laminar flow hood using a 10% sterile isotonic glucose solution (w/v) provided in thekit. This is required in order to form small and stable complexes. The use of ionicbuffers such as PBS or cell culture media for complex preparation should be avoided.
The final concentration of glucose in the injection volume should be 5 %.
 Prior to injections, ensure that Avalanche®-in vivo and glucose solution are equilibrated
at room temperature.
1.2 Protocol:
The amount of nucleic acids to deliver and Avalanche®-in vivo needed should bedetermined according to the animal model, the administration route and the targetedorgan. Recommendations for delivery of DNA, siRNA, oligonucleotides and shRNAexpressing plasmids in rodents are given in Table 1.

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

Dilute the nucleic acid using the 10% glucose stock solution (provided) and sterilewater to prepare a solution of ½ the injection volume of 5 % glucose. Vortex gently ormix by pipetting up and down.
Dilute the Avalanche®-in vivo using the 10% glucose stock solution (provided) andsterile water to prepare a solution of ½ the injection volume of 5 % glucose. Vortex
gently and spin down.
Add the diluted Avalanche®-in vivo to the diluted nucleic acid all at once, vortex gentlyand spin down.
Incubate for 15 minutes at room temperature. From this time point, the complexes
are stable 4 h at room temperature.
Perform injections into animals using complexes equilibrated at room temperature.
For siRNA and DNA immunization protocols, repeat injections up to 3 times a week if
required with freshly prepared complexes each time.

EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

2Avalanche®-in vivo Transfection Reagent
Monitor gene expression as required at the appropriate time point (6 – 72 h after thelast injection) depending on the mode of injection and the targeted organ.

1.3 Example: IV injection in mouse
Preparation of 300 μl injection volume of 5 % glucose containing 50 μg of plasmid DNA
and 10 μl of Avalanche®-in vivo
 Dilute 50 μg of DNA into 75 μl of 10% glucose; add sterile water to 150 μl, vortex
gently and spin down, Dilute 10 μl of Avalanche®-in vivo into 75 μl of 10% glucose; add sterile water to150 μl, vortex gently and spin down.
 Add the diluted Avalanche®-in vivo to the diluted DNA at once, vortex briefly andspin down.
 Incubate for 15 minutes at room temperature.
 Perform injections into animals using complexes equilibrated at roomtemperature.
 Monitor gene expression.
Table 1. Recommended conditions for most common injection routes in mice and rats
AnimalSite of injection

Complex formation

IV
Tail vein/retroorbital

50 (40-60) μg nucleic acid10.0 (6.0-14.0) μl reagent
in 300 μl of 5% glucose
100 (80-120) μg nucleic acid20.0 (12.0-28.0) μl reagent
in 1 ml 5% glucose
10 (8-12) μg nucleic acid
2.0 (1.2-2.8) μl reagent
in 50 μl of 5% glucose
5 (4-6) μg nucleic acid
1.0 (*0.6-1.4) μl reagent
in 10 μl of 5% glucose
1.5 (1.2-1.8) μg nucleic acid*0.3 (*0.2-*0.4) μl reagent
in 5 μl of 5% glucose
150 (120-180) μg nucleic acid
30 (18-42) μl reagent
in 1 ml of 5% glucose
3 (2.4-3.6) μg nucleic acid
0.6 (*0.4-0.8) μl reagent
in 10 μl of 5% glucose

IPMouseIntratumoral

Subcutaneous (s.c)

Intracerebral

IVRat

IntracerebralInjection volume(5% glucose)
300 μl

1 ml
50 μl
10 μl
5 μl

1.0 ml
10 μl

EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

3

Avalanche®-in vivo Transfection Reagent* Dilute Avalanche®-in vivo 1:4 with H2O prior to application (4 µl reagent + 12 µl H2O), andthen use 4 times of the volume in the table for accurate pipetting.
For siRNA delivery and DNA immunization protocols, multiple injections may be required.

TROUBLESHOOTING4Observations

Comments and Suggestions
Unsatisfactory resultsToxicity

Optimize the amount of nucleic acids used in the delivery
assay.
Use high quality plasmid or siRNA preparation. Ensure they
contain neither salt, RNA, protein or endotoxin. For plasmidDNA, OD260/280 ratio should be greater than 1.8. It is best
to use DNA prepared in water. For siRNA, prefer HPLC or
PAGE purified oligos.
Optimize Avalanche®-in vivo /nucleic acid ratio.
Check that the nucleic acid is efficient in vitro.
Ensure that the complexes are formed in glucose 5%.
Ensure that both nucleic acid and Avalanche®-in vivo are
diluted in 5% glucose before mixing.
Decrease the amount of nucleic acid, Avalanche®-in vivo, orboth.
If using plasmid DNA, ensure the preparation is endotoxinfree and in water.

Avalanche® is the trademark of EZ Biosystems LLC. All rights reserved.
Avalanche® series Transfection Reagents are sold to the Buyer with a limited license for Research Use
Only and is not for clinical, therapeutic or diagnostic use in humans or animals.
Avalanche® series Transfection Reagents may not be re-packaged or re-sold without written
permission from EZ Biosystems LLC. The buyer agrees not to attempt to reverse engineer,reconstruct, synthesize or otherwise modify EZ Biosystems Avalanche® products. A license from EZ
Biosystems LLC is required for commercial application of Avalanche® series Transfection Reagents.
For obtaining a license to use those products for commercial application, contact EZ Biosystems LLC
via business@ezbiosystems.com. For full terms and conditions, visit: www.ezbiosystems.com.

EZ Biosystems LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com