siRNA Encapsulation into Exosomes Using Electroporation: An Electric Pulse Based Technique to Load siRNA into Exosomes

Samenvatting

Source: Faruqu, F. N. et al. Preparation of Exosomes for siRNA Delivery to Cancer Cells. J. Vis. Exp. (2018)

This video describes an electroporation-based method for encapsulating siRNA into exosomes. The siRNA-loaded exosomes can then be used for therapeutic applications.

Protocol

1. siRNA Encapsulation into Exosomes by Electroporation

1. Pre-chill the electroporation cuvette (see Table of Materials) on ice for 30 min before electroporation.
2. Mix 7.0 µg of exosomes (32 µL from 7 x 10¹² p/mL stock in PBS) with 0.33 µg of siRNA (12 µL from 2 µM stock in RNase-free water) in the microcentrifuge tube. Make up the volume to 150 µL with citric acid buffer (see Table of Materials). The exosome to siRNA molar ratio is 1:60 in this case.
3. Transfer the mixture to electroporation cuvette. Cap the cuvette and place it in the cuvette holder....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Atto655-siRNA	Eurogentec	SQ-SIRNA	(Labelled-S) UGC-GCU-ACGAUC-GAC-GAU-G55; (UnlabelledAS) CAU-CGU-CGA-UCG-UAGCGC-A55.
Glass pipettes	Fisher Scientific	1156-6963
Amaxa Nucleofector I	Lonza	Nucleofector I	With Amaxa’s Nucleofector kits
Citric acid buffer with EDTA			Mix 0.1954 g citric acid and 0.2087 g disodium phosphate in 50 mL of deionised water. Add EDTA to 0.1 mM. Adjust pH to 4.4.
Microfuge tubes	Starlab	S1615-5500	1.5 ml