Exosome isolation from plasma using ExoQuick reagent

doi:10.1038/protex.2017.072

Method Article

Exosome isolation from plasma using ExoQuick reagent

https://doi.org/10.1038/protex.2017.072

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This protocol describes how to isolate exosomes from plasma using the ExoQuick Plasma Prep and Exosome Precipitation kit. It supercedes an earlier protocol (http://dx.doi.org/10.1038/protex.2015.108). The new protocol is based on our experience vetting the original protocol in multiple labs. The major change is an increase in input volume of biofluid from 200 µL to 500 µL. We found that the larger input volume led to more reproducible amounts of RNA isolation across multiple experiments. We have also included footnotes to explain changes we made to manufacturers' protocols, and observations that were made while carrying out the protocols.

Cell biology

Biological techniques

Molecular Biology

exRNA

extracellular vesicles

exosomes

plasma

blood

ExoQuick

Extracellular RNAs (exRNAs) have been found to play an important role in intercellular communication in the body. exRNAs are present in biofluids, in complexes with lipoproteins and ribonucleoproteins, and in extracellular vesicles such as exosomes and microvesicles. Further study and characterization of exRNAs and their carriers could lead to identification of new biomarkers and have potential for development of novel therapeutics.

ExoQuick Plasma Prep and Exosome Precipitation kit (System Biosciences – EXOQ5TM-1)

Phosphate-buffered saline (PBS)

Microfuge

1.5 mL Microfuge tubes

Transfer 500 µL of plasma into a 1.5 mL microfuge tube.
Add 5 µL thrombin (500U/mL) to a final concentration of 5U/mL.
Incubate at room temperature for 5 minutes while mixing (gently flicking tube).
Centrifuge at 10,000 x g for 5 minutes. There should be a visible fibrin pellet at the bottom of the tube.
Transfer supernatant to new microfuge tube.
Add 125 µL of ExoQuick Exosome Precipitation Solution to the plasma and incubate for 30 minutes at 4°C.
Centrifuge ExoQuick/plasma mixture at 1,500 × g for 30 minutes at room temperature.
Aspirate supernatant.
Spin down residual ExoQuick solution by centrifugation at 1,500 × g for 5 minutes at room temperature.
Remove all traces of fluid by aspiration, taking great care not to disturb the pellet.
Resuspend the pellet in 50 µL sterile PBS and proceed with RNA isolation using SeraMir Exosome RNA Purification Column Kit.

This protocol was modified from the manufacturer’s instructions for the ExoQuick Exosome Precipitation kit.

JF and AS were supported by NIH grant UH3 TR000943. PN, AP and RG were supported by UH2-UH3 TR000890. SS and LL were supported by U01 HL126494 and UH3 TR000906. XZ, LB and XOB were supported by U19 CA179463.

The authors declare no competing financial interests.

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Exosome isolation from plasma using ExoQuick reagent

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Exosome isolation from plasma using ExoQuick reagent

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Acknowledgements

Additional Declarations

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