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

Overview

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 of the electroporator (see Table of Materials). Rotate the turning wheel 180° clockwise.
   NOTE: The wheel must be turned completely to the locked position, in order for the cuvette to contact the electrodes.
4. Select the desired electroporation program (e.g., X-01, X-05, A-20, T-20, T-30, etc.) and start electroporation by pressing the start button.
   NOTE: A successful pulse is indicated by showing "OK" on the display.
5. Once electroporated, remove the cuvette after turning back the wheel 180° counterclockwise. Withdraw the sample from the cuvette with the plastic pipette for further processing.

Materials

Name	Company	Catalog Number	Comments
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