I am working on RNA isolation from exosomes of HEP3B and SNU398 3D spheroid cultured cells. I am getting around 100-200ng/ul RNA concentration, 260/280 of 2.00 from SNU398 cells but the HEP3B exosome RNA concentration is less than 5ng/ul or sometimes in negative with 260/280 of more than 3.00.
The methods of culturing and isolation of exosomes from these cells are similar and the particle count is also detected in a similar range from the NTA software for both of these cell lines. I have performed RNA isolation from the Qiagen kit and Trizol, both are giving the same odd value for HEP3B but everything is ok with SNU398 cells.
Does anyone have experience with these cell-derived exosomes, suggestions are highly appreciated.
Yes, I faced the same issue, but in my case, I worked with Danio oocytes—only about 100 oocytes in total. It was indeed challenging to extract RNA from such a small sample size.
Did you perform any DNase treatment after extracting the RNA? If so, I suggest trying to quantify the RNA without the DNase washing step. When dealing with very low RNA concentrations, the DNase treatment can lead to RNA loss, which might be causing the low yield issue.
Joyanta Bir Thank you for your answer. The way I am doing RNA isolation has no step of DNAase treatment.
I am planning to do the conventional phenol chloroform method.
Namrata Anand Good luck. In any case you can check my follwing method paper.
Article Method for the molecular and quantitative identification of ...
Hi Namrata Anand
I am trying to isolate exosomes from a spheroid model too. Did you use a kit for the exosome isolation? I'm currently looking for the best method to isolate them
Alejandra Toribio Valdez Hi Alejandra I have used Total Exosomes RNA isolation kit from Invitrogen. But I am getting RNA around 10-30ng/ul. It is not sufficient for our experiments. Can you tell me what kit or method you are using?
Thanks in Advance
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