Omics characterization of exosomes
EVerZom provides large characterization and quantification of exosomes services with OMICS approach.
In a first step, RNA must be extracted and purified from EV. The challenge is that RNA is present at a much lower level in EV than in cells. Obtained RNA can be analyzed by different methods to get the extraction yield. Electrophoresis provides information about RNA quality and size repartition. Concentration should be between 200 pg/ml and 1 µg/ml.
1 ng of small RNA is more than enough for small RNA-seq. Between 1 and 100 ng of total RNA is appropriate for RNA-seq. It will provide a list of present miRNA (normally 100-1000 miRNA detected) or mRNA, with the possibility of differential analysis between several samples. Biological triplicates provide robust estimation of miRNA quantitative presence and allows comparison between conditions.
Proteomic needs a purified protein samples, that can be extracted from isolated EV, cell supernatant, or cell lysate. There is a need of at least 10 µg of proteins to be processed and digested well before the analysis. It depends a lot on EV batch and process, but 10^11 EV would be large enough.
Proteomic detects and lists around a maximum of 2,000 proteins by EV sample, based on their peptide fragment analysis by mass spectrometry. It gives relative numbers that can serve for inter-sample comparison with a high precision, while intra-sample comparison between different proteins is more approximative. While the gold standard is biological replicate (N=3), it is also possible to replace it by technical replicate (n=3), with a lower confidence. Single measure would be enough to obtain a list of present proteins, but quantification is thus extremely approximative and statistical comparison impossible.
Depending on the scientific question, proteomics can provide important data: from the raw list of detected proteins, it is useful for the comparison between processes, producing cells, or batches providing specific signatures. Classification by protein family provides an overview of EV content. EV-associated protein detection confirms the nature of purified exosomes while impurities such as apolipoproteins, serum protein aggregates, cell compartments can be detected.
Due to their very low abundance, proteomics is not the method of choice for cytokines and growth factor quantification, compared to Luminex analysis.
Lipidomics is the quantitative analysis of lipid content of a sample. As biological lipids
are mostly distributed in 15-20 lipid classes (defined by their polar head), it
is possible to add internal standard representative of the most abundant
classes to allow precise and robust quantification of each lipid molecules
(around 200 different lipids are classically detected in biological samples).
Thus, lipidomics can give values in mol or g per volume unit in an absolute
manner, and give the percentage among total lipids for each one.
EV samples are optimally constituted from 5×1010 EV, in less than 200µL
(meaning [C]=2.5×1011 EV/mL of ammonium bicarbonate 150 mM buffer).
But it is possible to get some results from up to 5-10 time less EV content.
Thanks to lipids quantification, it is possible to know the percentage of lipids coming
for instance from lipoproteins (mainly constituted from cholesterol ester,
absent from cell membrane).
We use shotgun lipidomics (without preliminar chromatography), except if a better
sensitivity against precise lipid entities is required.