Did you confirm that you have the exosomes isolated and in enough amount? You can do this using Nanoparticle Tracking Analysis (NTA).

I isolated exosomes from MDA-231 cells using the same method as you did. Also, I tried a more reliable method using combined ultracentrifugation followed by ultrafiltration as follow:

Briefly, the conditioned medium was centrifuged at 600g for 30 min to remove cells and debris. The supernatant was centrifuged again at 2,000g for 30 min to remove apoptotic bodies. Microvesicles were collected by ultracentrifugation at 20,000g for 60 min.

Supernatants were filtered through 0.22- µm membrane filters (Thermo Scientific) with pressure to remove large vesicles.

Exosomes were collected by size-based EV isolation method with modifications [34] using 50 nm membrane filters (EMD Millipore, VMWP02500) with holders (EMD Millipore, SX0002500).

You can find the full protocol in our paper here

https://mct.aacrjournals.org/content/early/2019/08/28/1535-7163.MCT-19-0299

I will be happy to answer any more questions you have.

Thanks @SherifIbrahim. Actually I confirmed that I have vesicles both by NTA and by electron microscopy. Thats why I think its a problem with the quantity of vesicles secrted by MDA231 Cells. Maybe they have les RNA than other EVs

But thanks, I will check on that paper.