I would like to isolate total RNA from my cell lines's exosomes. My cells produce very low yields of exosomes. How much exosomes (microgram) at least (an estimation) do I need to have a total RNA yield which then I can make cDNAs for qRT-PCR studies? Could you mention a paper about it I can refer to? Of course it is difficult to estimate the yield since cell lines produce and release exosomes differentially. Thanks in advance.
Dear Alireza, qPCR is a method that allows you to amplify a single copy or a few copies of a piece of DNA. The extraordinariness of this method is that you don't need for huge quantities of sample, but you can amplify your DNA even starting from a single molecule of cDNA.
So, there is not a specific quantity of starting exosomes sample.
Best regards,
FM
Thanks Punith and Filippo for your comments. As far as I know, about 200 ng RNA is required for cDNA synthesis and of course it depends on how many reactions you want to have. It is important how much RNA yield you get out of exosomes. If you do not have enough exosomes, you will get low RNA yield for sure.
Dear Punith, the reference didn't give me the info! but thanks anyway.
it depends on other parts of your workflow, eg qRT-PCR conditions. Theoretically one RNA molecule is enough for amplification/detection right? in reality you might need more than that. The good starting volume for exosome isolation using ultra is 10-30 ml, and then- depending on the outcome- you can go up or down. with the Total exosome isolation reagent, we use 1 ml cell media input- as it recovers more exosomes