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1. Centrifuge solution at 4 °C at 1000 × g for 15 min.


2. Move supernatant to a new tube with sufficient additional vol-
   ume for 4× current volume.


3. Add 2 volumes of chilled (− 20 °C) absolute ethanol.


4. Invert tube several times to mix.


5. [Optional] Freeze tube in liquid nitrogen (see Note 3).


6. Centrifuge solution at 4 °C at 14,000 × g for 15 min.


7. Remove and discard supernatant.


8. Allow pellet to air dry or dry in vacuum dryer.


9. Resuspend pellet in sterile water or buffer as desired. DNA can
   be kept overnight at 37 °C to ensure complete resuspension.

Exosome isolation is a more nuanced process than isolation of

cfDNA. There is currently no complete consensus on what sizes

and classes of exosomes are most informative or useful for liquid

biopsy for cancer surveillance or management [ 13 , 14 ]. Thus, new

studies could reveal specific ways for isolation of exosome subpop-

ulations. These include the selection of specific filtration aliquots,

or use of target protein binding or other methods to isolate specific

exosome types. This method is intended to be broadly flexible,

utilizing spectroscopic detection of exosome bound proteins for

their detection in filtrate and not specifying aliquot numbers to be

retained. Operators should consider what sizes of exosomes they

wish to acquire and can adjust aliquot selection as desired.

In terms of contamination, similar considerations apply to this

method as to the isolation of cfDNA. These include contamination

by cells directly or the lysis of cells releasing their DNA. The isola-

tion of exosomes does mean the presence of proteins and cell

membrane fragments to contaminate the process, so a more aggres-

sive lysis/digestion step may be necessary compared to the cfDNA

method.

1. Collect blood in a vacutainer or similar vessel impregnated
   with EDTA, lithium heparin, or other anticoagulant.


2. Sufficient blood should be collected to maximize plasma yield,
   and hence overall DNA produced (see Note 1).


3. Immediately upon collection, gently invert collection tube five
   times to mix blood. Be careful not to shake or treat the blood
   violently in order to limit cell lysis.


4. Centrifuge blood at 2000 × g for 15–20 min, until plasma
   separates.


5. Using a pipette, transfer all available plasma to a new tube for
   protein digestion. Be careful not to disturb the cell layer inter-
   face (see Note 2).

3.1.3 DNA Purification

3.2 Isolation of DNA
from Exosomes

3.2.1 Plasma Separation

Robert A. Smith and Alfred K. Lam