Exosome Isolation to miRNA Extraction Protocol

Exosome Isolation from Conditioned Media

1. Collect conditioned medium.
2. Centrifuge at 500 g (RCF) for 10 min at 4 °C, with acceleration max (steady throughout the protocol) and deceleration max (until Step 9). It allows discarding live cells.
3. Remove the supernatant into new tubes. Without disturbing or touching the pellet, collect all the medium above the aforementioned line, and then pipette it into a new tube.
4. Repeat Steps 2 and 3.
5. Centrifuge at 2000 g for 15 min at 4 °C, and move the supernatant into new tubes. It allows to discard dead cells.
6. Repeat Step 5.
7. Centrifuge at 10,000 g for 30 min at 4 °C, and move the supernatant into new tubes. It allows to discard cell debris and large diameter vesicles.
8. Repeat Step 7.
9. Centrifuge the supernatant at 100,000 g for 1 h at 4 °C, reducing the braking power. If you are using new series machines (such as Optima XPN or XE), set deceleration at "5"; in case of previous series (such as Optima L), set at "slow brake".
10. Carefully remove most of the supernatant (always considering the light line before the round part of the tube) using large volume pipette while using 1 mL pipette upon reaching the pellet. Leave 2/3 mL supernatant for a better storage of your vesicles.
11. Add PBS to wash your exosome preparation to discard contaminants.
12. Centrifuge at 100,000 g for 1 h at 4 °C, reducing the braking power. If you are using new series machines, set on "10"; in case of previous series, set on "no brake".
13. Remove supernatant as for Step 10.
14. Resuspend the pellet in about 200 μL PBS.

Electron Microscopy

1. Starting from 200 μL exosome preparation, 25 μL are resuspended in 275 μL of 0,32% PBS citrate to reach a final volume of 300 μL.
2. Dilute 150 μL of exosome in 150 μL of 4% paraformaldehyde, thus reaching a final concentration of 2%. This step allows to fix your vesicles.
3. Incubate overnight at 4 °C.
4. Apply 10 μL of fixed vesicles to a carbon/formvar-coated EM grids for 7 mins.
5. Stain with 2% uranyl acetate for 30 min at RT.
6. Let the vesicles dehydrate for 2 h at RT.
7. Image with TEM at 100 kV.

Exo-miRNAs Content Analysis

1. Resuspend the exosome preparation in 1 mL TRIzol reagent. Shake vigorously, vortex, and store at −80 °C overnight.
2. Thaw samples at RT, add 200 μL 2-bromo-3-chloropropane (or chloroform), and vortex the tube vigorously. Incubate for 5 min at RT.
3. Centrifuge at 12,000 g for 15 min at 4 °C.
4. Transfer the aqueous phase to a new tube.
5. Add 500 μL cold isopropyl alcohol and vortex vigorously.
6. Freeze at −80 °C for 1 h.
7. Centrifuge at 12,000 g at 4 °C.
8. Remove isopropyl alcohol (as much as possible), and add in 1 mL cold 75% ethanol in DEPC-treated H2O.
9. Centrifuge at 7500 g for 5 min at 4 °C.
10. Remove ethanol (as much as possible).
11. Dry pellet at 42 °C until the pellet is dried.
12. Resuspend the pellet in 15 μL DEPC-treated H₂O (or RNase-free water).
13. Quantify extracted RNA using NanoDrop 1000 spectrophotometer.

RNA Quality Control

Gel Preparation

1. Transfer complete volume (about 650 μL) of Small RNA gel matrix into the top receptacle of a spin filter.
2. Place the spin filter in a microcentrifuge, and spin for 15 min at 10,000 g.
3. Remove the filter.
4. Store the filtered gel at 4 °C (until 1 month).

Gel-Dye Mix Preparation

1. Vortex Small RNA dye concentrate for 10 s and spin down.
2. Pipette 2 μL of dye into 0,5 mL RNase-free microtubes. Gel matrix is very viscous; thus avoid vortexing, and pipette slowly.
3. Add 40 μL of filtered gel.
4. Mix solution by pipetting or inverting the vial until the mix is homogeneous.
5. Spin tube at 13,000 g for 10 min at RT.

Gel-Dye Mix Loading

1. Take a new Small RNA chip out of its sealed bag.
2. Place the chip on the chip priming station.
3. Let the gel-dye mix equilibrate for 30 min at RT before use, protecting it from light.
4. Pipette 9 μL of the mix at the bottom of the well.
5. Set the timer at 60 s, and make sure that the plunger is positioned at 1 mL.
6. Close the chip priming station.
7. Press the plunger of the syringe down until it is held by the chip.
8. Wait for 60 s, and then release the plunger with the chip release mechanism.
9. Open the chip priming station, and pipette 9 μL of the gel-dye mix in each of the wells marked "G".

Small RNA Conditioning Solution and Marker Loading

1. Pipette 9 μL of the Small RNA conditioning solution into the well-marked "CS".
2. Pipette 5 μL of the marker into the well-marked with a ladder and each of the 11 sample wells. Do not leave any wells empty to allow a proper run of the chip. Add 5 μL marker +1 μL deionized water to each unused sample well.

Sample Loading

1. Heat denaturation of your sample at 70 °C for 2 min before loading on the chip.
2. Pipette 1 μL of the ladder into the well-marked with the ladder symbol.
3. Pipette 1 μL of each sample into each of the 11 sample wells.
4. Place the chip horizontally in the adapter of the vortex mixer, and vortex for 60 s at 2400 g.
5. Run your sample by the Agilent 2100 bioanalyzer within 5 min.

Reference:

1. Erika D A, Annamaria M, Giulia S, et al. Exosomal MicroRNAs: Comprehensive Methods from Exosome Isolation to miRNA Extraction and Purity Analysis[M]//MicroRNA Profiling. Humana, New York, NY, 2023: 75-92.