I am currently working with DLBCL cell lines for my project. I have isolated exosomes from DLBCL cell line using differential ultracentrifugation and ultrafiltration after which I went on to isolate RNA using RNeasy and miRVana kits, but the concentrations are in negative. Is there a better kit to perform this? What is the minimum volume of exosomes needed to perform an RNA isolation?
Here you could find video article on this subject:
J Vis Exp. 2012 Jan 9;(59):e3037. doi: 10.3791/3037. Isolation and characterization of RNA-containing exosomes. Lässer C1, Eldh M, Lötvall J.
https://www.jove.com/video/3037/isolation-and-characterization-of-rna-containing-exosomes
I personally found miRCURY RNA Isolation Kit works the best. Trizol is also OK but more hassle.
Thank you very much! We are actually growing our cells in medium containing FBS and then transferring them into exo-free FBS medium 48 hours before our isolation process. This is mainly due to the medium being very costly. But, is this process going to give us the right result? Or is there any other way we could optimize this? And, how is Total exosome isolation and Total exosomal RNA isolation reagent provided by Invitrogen life technologies? Any inputs on their working efficiency?
Hi Sneha,
While you intend to perform RNA isolation from Exosomes (or other microvesicles) for downstream analysis (including RNA functional analysis or RNA profiling), you need to keep in view that a fraction of RNA comes from FBS. Even though you have performed exosome depletion steps for FBS, it is not always depleted 100% even after after extended steps of ultracentrifugation. For this, you may wish to consult this recent article in Sci Reports by Wei et al.
https://www.nature.com/articles/srep31175
Thank you very much for providing this article! I will go through this and see if I can troubleshoot any of my steps to make things work.
in extension to my previous answer, you may also wish to consider this article by Victoria El-Khoury https://www.nature.com/articles/srep19529 .
Regarding, your query about best way of isolating RNA from cell culture, miRCURY Isolation kit for cell cultures has been proven a best one in terms of RNA recovery from cell culture derived exosomes. For this you can see an article in which different6 methods have been compared. it will give you a possible answer to your question.
see link here.
https://www.ncbi.nlm.nih.gov/pubmed/?term=22424315
Good luck
We actually tried working with exosomes by transferring the cell lines into exo-depleted FBS medium 48 hours before our isolation process, but we neither got a good concentration nor did we identify anything on the Western blot analysis. We now started growing our cells in exo-free FBS right from the beginning and planning to use Total exosome isolation reagent by Invitrogen. Any idea on how it works? I have read articles that have used that kit to isolate exosomes and exosomal RNA and have attained good yields.
Before using a general precipitation approach in the early phases of a project, consult the following:
http://www.tandfonline.com/doi/full/10.3402/jev.v3.26913
https://www.ncbi.nlm.nih.gov/pubmed/28245209
Kit-based approaches that involve polyethylene glycol (PEG) precipitation can be very easy and useful for concentrating large volumes or even for preparing particles after the active component has already been rigorously shown to be exosomes/other EVs. However, for discovery projects focused on EVs, salting out by PEG will precipitate everything in the fluid of interest, and only a minority of RNA is associated with EVs in most systems. Without subsequent purification steps and rigorous characterization, it will be difficult or impossible to attribute any observations to EVs. Residual PEG may also interfere with subsequent assays.
As for nucleic acid concentrations, one will find only very low concentrations of EV RNA in most systems, especially if the chosen separation method is not rapid and efficient. qPCR for ubiquitous RNAs (such as abundant miRNAs) may be more informative than total RNA measurements, again with the caveat that purity measurements are needed: positive and negative EV markers.
Additionally, I'd advise to perform a gradient centrifugation step during EV purification.
As Kenneth noted, many other RNA-containing structures are present in pellets obtained from culture supernatant which might obscure your analyses.
I've isolated RNA from floating EV-samples together with non-floating samples and found a considerable amount of RNA in the non-floating fractions. These non-floating samples had a different RNA composition than the floating EV-samples, and were less resistant to RNAse/protease treatment, suggesting these are different structures.
Thanks for the suggestion Tom! I just wanted to clarify, will this be the case even when performing isolation of exosomes using the kit? I just read a paper that said, sucrose gradient would be effective to purify exosomes that underwent the differential ultracentrifugation. And, I have been seeing multiple protocols for performing a sucrose gradient centrifugation. Can you suggest an optimized protocol to work with if needed?
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