Hiii Researchers,
I have recently started working on exosomes and curious to explore more in this field. Unlike cell lysates where we use housekeeping gene expressions as normalization control, what should we use in case of exosomes? I'm not sure about how we can determine differentially expressed protein in exosomes? Also, if someone can explain about how to determine the upregulated or downregulated microRNAs or mRNAs in the exosomes? How to normalize? It would be a great help if someone can share their valuable inputs. Looking forward to a vital response from the scientific community.
Thanks.
In my opinion and experience, the exosomes particles concentration is a normalization control. Exosomes particle concentration could be determined by some techniques e.g. NTA, and Nano-flow. Therefore, you can calculate how many protein/RNA copies in one particle on average. On the other hand, if you want to chose some control like doing Western-blot, some exosomes marker protein such as Tsg101, Alix, Syntenin, CD63 etc, may be selected. However, please remember the abundance of these marker proteins could be altered by the over-expression of your interested genes.
There are two levels of comparative analysis you can perform,
1. Exosomes vs parental cells. You can normalize based on total proteins (i.e. ug to ug). for westerns, you could potentially use GAPDH, although recently exosomes were reported to lack GAPDH proteins. But EVs in general are reported to carry GAPDH
2. Exosomes from one condition vs another condition. here you can also normalise based on total proteins. for western you can use EV markers such as ALIX
for RNA comparison, you can follow similar concept or normalize based on RNA amount (although EVs are thought to be rich in RNA per ug of protein )
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