Exosomes can be derived from ultracentrifugation or isolated by exosome isolation kit, such as Exosome Isolation Kit (from cell culture medium, Cat. No.: EZB-exo2).
Exosome samples are first lysed in a strong denaturant and phenol containing buffer, which immediately inactivates RNases to ensure isolation of intact total RNA. After sample lysis, chloroform is mixed with the lysate and incubated for 3 minutes. And then the mixture is centrifuged for phase separation; the upper-layer supernatant is collected and mixed with ethanol. The liquid is then passed through the RNA binding Spin Column containing a silica-based membrane to which the RNA binds. Impurities are effectively removed by subsequent washing. The purified total RNA is then eluted with Elution Buffer and may be used in a variety of downstream applications.
And the isolated exosomes can be used for RNA purification and detection, such as RT-qPCR, RNA sequencing, RNA Chip, and so on.
Components
Component | EZB-exo-RN1 (50 Preps) |
Lysis Buffer | 30 ml |
Wash Buffer 1* | 8 ml |
Wash Buffer 2* | 8 ml |
Elution Buffer | 25 ml |
Spin Columns (with Collection Tubes) | 50 Preps |
*Before using for the first time, add 32 ml of 100% ethanol to the Wash Buffer 1, 32 ml of 100% ethanol to the Wash Buffer 2.
Storage
Store the Lysis Buffer at 2 ~ 8°C, protect from light. Store other components at room temperature (When using these buffers, be careful to avoid of contamination). Divide the Elution Buffer into small aliquots upon reception is suggested.
Experimental Procedure at a Glance
Protocol
Sample Lysis
1. Place 10 ~ 100μl exosomes in a 1.5 ml centrifuge tube. Add 500 μl of Lysis Buffer. (Exosomes purified from more than 0.5 ml of serum/plasma, or more than 10 ml of cell culture medium is recommended, for each preparation).
2. Lyse the exosomes by pipette up and down for 10 times. Incubate at room temperature for 5 minutes.
3. Add 100 μl chloroform to the exosome lysate, mix by pipette and hand-shaking. Incubate at room temperature for 3 minutes.
4. Centrifuge at 12000 × g, 4 °C for 2 minutes. Then the mixture separates into three phases. Transfer the upper-layer supernatant (about 300 μl) to a new RNase free 1.5 ml centrifuge tube (be careful to avoid disturbing the middle or bottom layer).
RNA Binding
5. Add 1.6 volume of 100% ethanol to each volume of supernatant for microRNA included total RNA purification.
6. Invert the centrifuge tube for several times or pipette up and down for 10 times to mix thoroughly, and transfer the sample to the Spin Column. Centrifuge at 4000 × g, 4 °C for 1 minute. Pour off the liquid.
Column Washing
7. Add 500 μl of Wash Buffer 1 to the column. Centrifuge at 12000 × g, 4 °C for 1 minute (be careful to avoid of contacting the bottom of the column with the liquid when taking out of the column). Pour off the liquid.
8. Add 500 μl of Wash Buffer 2 to the column. Centrifuge at 12000 × g, 4 °C for 1 minute.
9. Pour off the liquid and eliminate the residual liquid using towel paper. Place the empty column back on the collection tube and Centrifuge at 12000 × g, 4 °C for 1 minute.
10. Transfer the column to an RNase free 1.5 ml centrifuge tube, open the lid and keep in the air for 2 minutes.
RNA Elution
11. Add 20 ~ 30 μl of Elution Buffer to the center of the column and incubate at room temperature for 2 minutes.
12. Centrifuge at 12000 × g, 4 °C for 1 minute (transfer the eluate back to the column, incubate for 5 minutes and centrifuge once more will get more RNA).
13. Discard the column，do the following experiment with the purified RNA, or store the RNA at -80 °C until needed.
Note:
The concentrations of the most exosome RNAs are always too low to be measured by common microscale ultraviolet spectrophotometer, such as Nanodrop. If you need to measure the exosome RNA concentration, the Qubit (from Thermo Fisher) is suggested. In most experiments, the measurement of the exosome RNA concentration is unnecessary, such as qRT-PCR. You just need to make sure to use exosomes isolated from equal samples.
For detailed information, please check the product manual.
