Exosome Isolation: A Technique to Separate Exosomes from the Plasma of Non-small Cell Lung Cancer Patients
Transcript
– Exosomes are small, membrane-bound vesicles carrying biomolecules like lipids, proteins, DNA, mRNA, and microRNA. Exosomes are released by different cell types into extracellular spaces and can be isolated from biofluids like plasma.
To begin, centrifuge the plasma sample to remove cells and debris. Transfer the plasma supernatant into a fresh tube. Treat the plasma with the desired concentration of ribonuclease enzyme. Incubate the sample on a heating block at 37 degrees Celsius for an appropriate duration.
The enzyme degrades any free RNA in the sample, while the exosomal RNA remains protected. Dilute the sample using PBS. Next, add the desired concentration of Proteinase K enzyme solution and incubate at 37 degrees Celsius for an appropriate time. Proteinase K degrades the bulk of plasma proteins. It also inactivates the previously added ribonuclease enzyme, thereby eliminating the risk of exosomal RNA degradation during downstream processing.
Add a suitable volume of exosome precipitation buffer to facilitate exosome separation from the plasma. Centrifuge the sample to obtain a pellet containing intact exosomes. The isolated exosomes find applications in downstream steps like RNA and protein extractions.
In the following protocol, we will perform exosome isolation from the plasma sample of non-small cell lung cancer patients.
– To begin, thaw a 1 milliliter plasma sample on ice. Once thawed, invert the tube several times to disaggregate any cryoprecipitates that may have formed. Then, centrifuge the plasma at 2,000 Gs for 20 minutes at room temperature, in order to remove cells and debris. Collect the supernatant and centrifuge the plasma again, now at 10,000 Gs for 20 minutes. Again, collect the plasma supernatant and now add 10 microliters of RNAse. Incubate the plasma at 37 degrees Celsius for 10 minutes in a thermal block heater.
– We added an RNAse treatment step to the standard kit protocol in order to degrade the free microRNAs. Later, the proteinase K treatment step, included in the kit protocol was exploited in order to degrade the RNAse and protect the integrity of exosomal microRNAs.
– Next, transfer the plasma to a new tube at half a volume of 1x PBS and vortex it. Then, add 0.05 volumes of proteinase K solution, vortex the sample again and incubate it at 37 degrees Celsius for 10 minutes. Now, add 0.2 volumes of exosome precipitation buffer and mix the sample by inversion. Then, incubate the sample at four degrees Celsius for 30 minutes.
After the cold incubation, centrifuge the sample at 10,000 Gs for five minutes at room temperature. Take a 20 to 50 microliters aliquot of the supernatant and store it for control purposes. Then aspirate and discard the remaining supernatant.
Resuspend the exosome pellet in the buffer of choice, at a volume depending on the downstream analysis. For RNA analysis, add specific lysis buffer for RNA extraction.
