Overview
The plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of the biomarkers for early detection and disease progression. This video describes the protocol for the separation of exosomes from the plasma samples of non-small cell lung cancer patients, which could be used for DNA and protein extractions.
Protocol
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).
- Take 1 ml of plasma of each sample, stored at -80 °C, and place it on ice.
- Centrifuge the sample at 2,000 × g for 20 min at RT; this passage is mandatory in order to remove cells and debris.
- Transfer the supernatant containing the clean plasma to a new 1.5 ml tube.
- Centrifuge the new tube at 10,000 × g for 20 min at RT.
- Transfer the supernatant containing the clean plasma to a new 1.5 ml tube.
- 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.
- Transfer all the treated plasma to a new 2 ml tube and add 0.5 volume of 1x PBS.
- Vortex the sample in order to mix the solutions.
- Add 0.05 volumes of Proteinase K solution (enclose in the kit) to the sample.
- Vortex the sample and then incubate at 37 °C for 10 min.
- Add 0.2 volume of exosome precipitation buffer to the sample and mix the solutions by inversion.
- 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.
- Aspirate and discard the supernatant. The pellet contains the isolated exosomes.
- 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:
- Add 100 µl of 1x PBS in order to perform TEM analysis.
- Add 346.5 µl of specific lysis buffer for RNA extraction (from commercial kit) (CAUTION: chemical hazard).
- 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).
- Store the resuspended exosomes at −20 °C.
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Materials
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 |