193- Centrifuge isolated plasma at 4 °C at 1000 × g for 10 min fol-
lowed by centrifugation at 4 °C at 10,000 × g for 30 min. - Remove supernatant by pipette and pass through a 0.22 μm
filter. - Apply 0.5 mL of filtrate to a chromatographic separation col-
umn packed with Sepharose 2B and equilibrated with phos-
phate buffered saline (PBS). Passage aliquot through column
using 9 mL phosphate buffered saline. - Collect 1 mL aliquots of separated filtrate.
- Subject aliquots to spectrophotometry at 280 nm absorbance
and select aliquots with the highest absorbance for further pro-
cessing. Discard other aliquots. - Subject aliquots to ultracentrifugation at 4 °C at 105,000 × g
for 2 h. - Remove supernatant and discard.
- Resuspend pellet in 1 mL phosphate buffered saline.
- Add extracted plasma to a buffer solution for exosomes.
- Add proteinase K to a concentration of 50–100 μg/mL.
- Incubate solution in an agitating water bath at 37–56 °C
overnight. - [Optional] Incubate solution at 65 °C for 10 min to inactivate
proteinase K. - Centrifuge solution at 4 °C at 1000 × g for 15 min.
- Move supernatant to a new tube.
- Centrifuge solution at 4 °C at 1000 × g for 15 min.
- Move supernatant to a new tube with sufficient additional vol-
ume for 4× current volume. - Add 2 volumes of chilled (− 20 °C) absolute ethanol.
- Invert tube several times to mix.
- [Optional] Freeze tube in liquid nitrogen (see Note 3).
- Centrifuge solution at 4 °C at 14,000 × g for 15 min.
- Remove and discard supernatant.
- Allow pellet to air dry or dry in vacuum dryer.
- Resuspend pellet in sterile water or buffer as desired. DNA can
be kept overnight at 37 °C to ensure complete resuspension.
3.2.2 Exosome Isolation
3.2.3 Proteinase K
Digestion and Exosome
Lysis
3.2.4 DNA Purification
Cell-Free Plasma and Exosome-Associated DNA