Altogen Nanoparticle In Vivo Transfection Reagent

November 05, 2015

University
Pathology
PhD student

Overall

Quality of Results

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Company:

Altogen Biosystems

Product Name:

NANOPARTICLE In Vivo Transfection Reagent

Catalog Number:

5030

We have successfully used this reagent for siRNA. With siRNA, we were able to achieve 90% knockdown of the target gene, GAPDH in tissue lysate samples in BALB/C mouse, reproducibly. Furthermore, we did not see significant toxicity effects of the reagent after injection. A simple and easy to use protocol is supplied by the Altogen Biosystems kit which helped the successful outcome of experiment. Finally, the cost of the reagents from Altogen Biosystems is inexpensive compared to other sources.

Experimental Design and Results Summary

Application

siRNA and plasmid DNA delivery via direct subcutaneous tumor injection

Starting Material

The nanoparticle conjugated complexes are stable in serum for at least 16 hours, this facilitates for the efficient invivo delivery in to various organs. Inflammatory response due to the administration of Altogen Biosystems Nanoparticle Transfection Reagent is negligible.

Protocol Overview

The following protocol is used to knockdown GAPDH in BALB/C mouse. 100 µg of siRNA is diluted in 50 µl RNase-/DNase-free water and vortexed in the next step 50 µl of diluted siRNA is added to the sterile tube containing 50 µl Transfection Reagent. This mixture is incubated for 15-20 minutes at room temperature. In the later step 10 µl of Transfection Enhancer Reagent supplied with Altogen Bio systems kit is added and vortexed gently. This solution is incubated again for 5 minutes at room temperature and injected in to BALB/C mouse. Total protein was assessed using Thermofisher/Ambion PARIS protein isolation kit following manufacturer recommended protocol. Tissues were collected and RNA is isolated 48 hours after first injection. Samples were analyzed by qRT-PCR for Lamin A/C gene expression levels. GAPDH was measured 48 hrs post injection of nanoparticle invivo transfection reagent. Samples were analyzed on Wes run using primary antibody diluted 1:10 and secondary antibody diluted 1:25. 4µL of each sample was used for analysis. The total protein concentrations for all samples were normalized to 250 µg/mL.

Tips

Following the simple and easy to use protocol supplied by the Altogen Biosystems kit helps in the successful outcome of experiment.

Results Summary

Western blot of tissue lysates using GAPDH Antibody [HRP] are shown in the image. Western Blot was performed using Wes system (Manufacturer Protein Simple). The GAPDH protein was observed at ~40 kDa as expected.

Additional Notes

None

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Summary

The Good

It is possible to use the Altogen Biosystems Nanoparticle Transfection Reagent through various administration routes and has the capacity to form stable complexes with nucleic acid. We have successfully used this reagent for siRNA. With siRNA, we were able to achieve 90% knockdown of the target gene, GAPDH in tissue lysate samples in BALB/C mouse, reproducibly. Furthermore, we did not see significant toxicity effects of the reagent after injection. A simple and easy to use protocol is supplied by the Altogen Biosystems kit helped the successful outcome of experiment. Finally, the cost of the reagents from Altogen Biosystems is inexpensive compared to other sources.

The Bad

We have not observed any drawbacks that would interfere with the experiment.

The Bottom Line

Altogen Biosystems Nanoparticle Transfection Reagent is a highly successful nanoparticle-based reagent

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