Hello to everyone,
I'm trying to extrtact total RNA from sEVs.
I made a first experiment starting from 2x10^10 sEVs quantified by NTA but the RNA obtained was very low (under 20ng/ml)
Do you have some experience with this? Can someone give to me some suggestion?
Thanks
ask this of your sequencing core/provider. See if you can just send them the entire amount you isolate and keep just a small amount for NTA. Our core can use a low input kit on 10ng RNA so your samples might be usable.
are these from cell media supernatant? if so then plan to collect A LOT of cell supernatant. using T75 or 10cm dishes if possible for conditions. collect every 24h as EVs will get reabsorbed then extract as you have been. concentrate the media with 100KD MWCO centrifugation filters if needed/possible then isolate. Also look at your isolation method. I got much more EVs recovered with the Takara columns (however a search of their site shows they might be discontinued or bought out, but they were called Capturem extracellular vesicle isolation kits, you could try emailing them to see if they can send some or if they renamed it) than with ultracentrifugation - the pellet was very sticky and hard to get in its entirety. I've also used ExoQuick and that worked well too. they will send out small samples if you ask.
I dont think that 20 ng/ul is a low concentration for exosomal RNA. Since exosomal RNA is mainly small RNA, it will never give you very high concentrations. I did miRNA sequencing from lower concentrations. Some companies require a total amount of >10 ng of RNA for miRNA sequencing. However, important is that your RNA sample contains enough portion of small RNA (20-150 bp), thats where the miRNAs are.
Lívia Kolesár Fecskeová thank you for your answer.
I had a concentration of 1 ng/ul, so I'm trying to increase cell number from which I solate exosomes.
From how much cells did you start?
What kind of miRNA analysis do you want to do? If you aren't looking for a low copy miRNA then 1ng/ul is more than enough for RT-qPCR. I have collaborators who did RNA sequencing on RNA isolated from exosomes and they had success with RNA concentrations lower than 1ng/ul.
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