Hi everybody, I looking for a very good method to isolate exosomes from serum and urine. I will use these exosomes from study microRNAs. I'm never have worked with exosomes before so, I don't have experience in this area. I hope you can share me your experiences about this issue.
Thanks.
Hello, i would recommend taking a look at Qiagen's products.
https://www.qiagen.com/us/qdm/rna/exorneasy/poster/
Hi Marian,
welcome to the wonderful world of exosomes and EVs in general!
First of all, please be aware that the “best” method for serum might not be the most appropriate one for urine as well.
Next, defining a “best” method for EV isolation depends heavily on your respective requirements. Things like “how pure do the EVs need to be”, “what’s the downstream application”, “how much biofluid is available”, “are there limits on time, costs and equipment” all influence how suitable a given method is.
In our lab, we have screened quite a few methods for EV isolation from serum, and all of them have their pros and cons. As we’re also interested in miRNAs, we’ve found precipitation-based isolation (i.e. miRCURY, ExoQuick, TEI) to be very suitable. Isolation is fast, robust, pulls down a lot of RNA and results in reliable, high-quality sequencing libraries. At the same time, however, we know that the majority of material/RNA capture by precipitation is not associated to EVs at all – the method should be considered as unspecific ally enriching cell-free material. If that’s ok for your application (i.e. your samples don’t necessarily have to be pure exosomes), you could give one of the kits mentioned above a try.
On the opposite end of the spectrum there are methods such as density gradient centrifugation or size-exclusion chromatography, providing higher purity at the cost of markedly reduced material captured by isolation. The Qiagen kit mentioned by Can might be a good compromise; we’ve found it to be a little more specific than precipitation (pulls down less soluble protein) while still isolating enough material to generate good libraries (which still also contain lots of non-EV sequences, that’s for sure).
So long story short: you need to be very specific about your requirements before embarking on the journey to pick the most suitable isolation method!
Good luck,
Dominik
Hi Dominik, your answer was very complete! I appreciate.
What happen if I want to purify these exosomes isolated?
Do you think I can use some precipitation-based method and then use other technique for purify/characterize my exosome population?
Hi Marian,
sure, that's definitely feasible! For instance, we've used both density gradients and size-exclusion columns to further purify EVs initially isolated by precipitation.
Each of these methods has its pros and cons (and mode of action), but both are pretty effective at removing a bunch of contaminants. For ultra-pure EV preparations, a combination of methods based on both size and density would yield even better results.
For precipitated serum EVs, we've found that running an additional SEC purification step removes a huge amount of soluble protein (and RNA, unfortunately).
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