Hi,
I am relatively new to the exosome/MV study.
I would like to know which proteins and RNAs are absent (or likely to be absent) in exosomes/MV (negative control). I am aware about the proteins and RNAs present in exosomes (positive control).
In short, I would like to know if my isolated exosome fraction is pure and not contaminated with any other RNA or proteins. TEM would be my next step but before I would like to know the purity by observing protein and RNAs which are absent in exosome.
Any help is appreciated.
Thank you.
Commonly used non-exosomal markers are calnexin (CANX), nucleoporin, GM130, and Bcl-2.
Currently there is no consensus about a general exosome biomarker. The current recommendation is to combine detection of many membrane-bound or membrane-associated proteins in order to verify the presence of membranes.
Many exosomes contain proteins that are common among all exosomes regardless of the types of cells which secrete them. Top ten proteins found in the exosomes include Heat shock protein 8 (HSPA8), CD63 antigen (CD63), Actin, β (ACTB), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Enolase 1, α (ENO1), Heat shock protein 90, α (cytosolic), class A member 1 (HSP90AA1), CD9 antigen (CD9), CD81 antigen (CD81), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), Pyruvate kinase, muscle (PKM2). Tetraspanins are common exosome specific markers. They include CD9, CD63 and CD81 membrane proteins which are good combination for exosome marker.
However, a small fraction of proteins and RNAs are cell-specific, reflecting the type and pathophysiological conditions of the secreting cells. You should take them into account.
I would recommend reading:
Progress in Exosome Isolation Techniques
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327650/
Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548045/
Exosomes: isolation and characterization methods and specific markers
https://www.labome.com/method/Exosomes-isolation-and-characterization-methods-and-specific-markers.html
Thank you Dimitar Parvanov. Your answer is helpful. Sorry for asking again but beside proteins, are there any mRNAs or nuclear RNA which should be absent in Exosomes? I am mindful about RNA content in exosome which varies based on the type of cell they originate from.
Hello Dr.Kamlesh
I'll go off the tangent a bit and ask you, what RNA do you take as a positive control? I have been looking in existing literature but can't find any such reference. It'd really help if you could provide that.
As for the negative control, I think rRNA shouldn't be found in exosomal RNA preps. Ref: Article Isolation and Characterization of RNA-Containing Exosomes
Thank you Nishtha for adding answer.
Clarification on rRNA in exosomes.
http://www.pnas.org/content/114/43/E8987.long
To answer you question, It depend upon the cell type that I am using. I would search (good) papers who used same cells to study RNA in exosomes. Based on the search, I would take at least 2-3 RNAs as a positive control. Eg. Two papers (published in Nature/Cell) use "X" cell for their exosome study and both paper found "A" and "B" RNA abundant in exosomes, then I will used "A" and "B" as a positive marker for my study quoting those papers.
I hope this answers your question.
It totally depends from which cell type you are isolating the vesicles.
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