Exosome Isolation: A Technique to Separate Exosomes from the Plasma of Non-small Cell Lung Cancer Patients

プロトコル

1. Exosome Isolation

Note: The isolation of exosomes from plasma samples, was performed with a commercial kit, according to manufacturer's protocol and by adding RNAse treatment: (all these steps must be performed with personal and environment protective equipments and following specific legal procedure for manipulation of biological samples).

1. Take 1 ml of plasma of each sample, stored at -80 °C, and place it on ice.
2. Centrifuge the sample at 2,000 × g for 20 min at RT; this passage is mandatory in order to remove cells and debris.
3. Transfer the supernatant containing the clean plasma to a new 1.5 ml tube.
4. Centrifuge the new tube at 10,000 × g for 20 min at RT.
5. Transfer the supernatant containing the clean plasma to a new 1.5 ml tube.
6. Add 100 ng/ml of RNAse to the supernatant and incubate the tube at 37 °C for 10 min in order to degrade circulating RNAs.
7. Transfer all the treated plasma to a new 2 ml tube and add 0.5 volume of 1x PBS.
8. Vortex the sample in order to mix the solutions.
9. Add 0.05 volumes of Proteinase K solution (enclose in the kit) to the sample.
10. Vortex the sample and then incubate at 37 °C for 10 min.
11. Add 0.2 volume of exosome precipitation buffer to the sample and mix the solutions by inversion.
12. Incubate the sample at 2 °C to 8 °C for 30 min and then centrifuge the sample at 10,000 × g for 5 min at RT.
13. Aspirate and discard the supernatant. The pellet contains the isolated exosomes.
14. Add selected buffer to the exosome pellet in order to resuspend the exosomes. The selection of the buffer depends on the downstream analysis planned, in this case:
    1. Add 100 µl of 1x PBS in order to perform TEM analysis.
    2. Add 346.5 µl of specific lysis buffer for RNA extraction (from commercial kit) (CAUTION: chemical hazard).
    3. Add 100 µl of lysis buffer for protein extraction (Tris-HCl 50 mM pH 7.6 - NaCl 300 mM - Triton X-100 0.5% - PMSF 1 mM - Leupeptin/Aprotinin 10 µg/ml) in order to perform Western Blotting analysis. (CAUTION: chemical hazards).
15. Store the resuspended exosomes at −20 °C.

資料

Name	Company	Catalog Number	Comments
Total exosome isolation kit (from plasma)	Invitrogen	4484450	Use Proteinase K treatment
Ribonuclease A	Sigma-Aldrich	R4875-100MG
RNAspin Illustra mini kit 50	GE HealthCare	25-0500-71