32
4. Remove the excess water using filter paper.
5. Transfer the grids onto 1% glutaraldehyde drop placed on
parafilm and leave the exosomes for further fixation for a period
of 5 min.
6. Repeat the washing step three times and remove the excess
water using filter paper.
7. Put the grids onto 0.5% uranyl acetate drops and incubate for
10 min for adequate staining (see Note 3).
8. Observe the samples under the electron microscope (Joel USA
JEM 2100-F).
1. Put 175 μL RNA lysis buffer into an Eppendorf tube in which
30 mg of exosome suspension is situated.
2. Following homogenization by vortexing, pipetting is done for
efficient lysis.
3. Add 350 μL of RNA dilution buffer to 175 μL of lysate. Mix
by inverting the tube 3–4 times. Place in a heating block at
70 °C for 3 min.
4. Centrifuge at 13.000 × g for 10 min at 20–25 °C.
5. Transfer the lysate into a clean tube and add 200 μL 95% ethanol. Mix by pipetting 3–4 times.
6. Transfer the mixture to the spin column assembly and centrifuge at 13,000 × g for 1 min.
7. Discard the liquid in the collection tube and add 600 μL of
RNA wash solution to the spin column assembly. Centrifuge
at 13,000 × g for 1 min.
8. Apply 50 μL of the DNase incubation mix onto the column
membrane and incubate for 15 min at room temperature. Add
200 μL of DNase stop solution and centrifuge at 13,000 × g
for 1 min.
9. Add 600 μL of wash solution and centrifuge at 13,000 × g for
1 min.
10. Discard the liquid in the collection tube and add 250 μL RNA
wash solution into the column. Centrifuge at 13,000 × g for
2 min.
11. Add 100 μL of nuclease-free water into the column fitted with
an elution tube and centrifuge for 1 min.
12. Quantify the amount of exosomal RNA using a Nanodrop
(Thermo Fisher) by dropping 5 μL of RNA solution onto the
pedestal (see Note 4).
3.6 RNA Isolation
and Quantification
Burak Derkus and Emel Emregul
4. Remove the excess water using filter paper.
5. Transfer the grids onto 1% glutaraldehyde drop placed on
parafilm and leave the exosomes for further fixation for a period
of 5 min.
6. Repeat the washing step three times and remove the excess
water using filter paper.
7. Put the grids onto 0.5% uranyl acetate drops and incubate for
10 min for adequate staining (see Note 3).
8. Observe the samples under the electron microscope (Joel USA
JEM 2100-F).
1. Put 175 μL RNA lysis buffer into an Eppendorf tube in which
30 mg of exosome suspension is situated.
2. Following homogenization by vortexing, pipetting is done for
efficient lysis.
3. Add 350 μL of RNA dilution buffer to 175 μL of lysate. Mix
by inverting the tube 3–4 times. Place in a heating block at
70 °C for 3 min.
4. Centrifuge at 13.000 × g for 10 min at 20–25 °C.
5. Transfer the lysate into a clean tube and add 200 μL 95% ethanol. Mix by pipetting 3–4 times.
6. Transfer the mixture to the spin column assembly and centrifuge at 13,000 × g for 1 min.
7. Discard the liquid in the collection tube and add 600 μL of
RNA wash solution to the spin column assembly. Centrifuge
at 13,000 × g for 1 min.
8. Apply 50 μL of the DNase incubation mix onto the column
membrane and incubate for 15 min at room temperature. Add
200 μL of DNase stop solution and centrifuge at 13,000 × g
for 1 min.
9. Add 600 μL of wash solution and centrifuge at 13,000 × g for
1 min.
10. Discard the liquid in the collection tube and add 250 μL RNA
wash solution into the column. Centrifuge at 13,000 × g for
2 min.
11. Add 100 μL of nuclease-free water into the column fitted with
an elution tube and centrifuge for 1 min.
12. Quantify the amount of exosomal RNA using a Nanodrop
(Thermo Fisher) by dropping 5 μL of RNA solution onto the
pedestal (see Note 4).
3.6 RNA Isolation
and Quantification
Burak Derkus and Emel Emregul