Hello,
I have been trying to isolate RNA from EVs (from MSCs) for a while. I isolate the EVs using SEC. Then I use the "Total Exosome RNA and Protein Isolation Kit" from Invitrogen. When I measure the RNA with the nanordop, I only get a peak at 250 nm, which indicates contamination with guanidinium isocyanate.
I have also measured the RNA with the "Qubit fluorometer", but the concentration is not in the measurable range (under 20 ng/µl).
I checked the quality and intigrity with the bioanalyzer, it showed that the RNA is completely degraded and there is hardly any RNA in the samples.
Does anyone have experience with RNA isolation and can help me?
Thanks a lot!
Hi Samira,
I assume that the degradation occurred due to 2 reasons: incorrect storage during collection, or during extraction. If it is occurred during storage, you need to make sure that your samples are frozen immediately after collection. In this case you can use -80 or liquid nitrogen. While if the degradation happened during extraction, try to add betamercaptoethanol (BME) to the lysis buffer. Use 10 µl of 14.3 M BME per 1 ml of lysis buffer. BME kills the RNases and helps in stabilizing the sample during extraction. For more information, check this out https://bitesizebio.com/2345/troubleshooting-rna-isolation/
I think the overall RNAs from EV is relatively low, you would need harvest and concentrated.
Hello,
Your RNA is not degraded. I worked with RNA from EV and I have the same results. You can'nt mesure your RNA with nanodrop because it is very low (pg/µl) for this reason, you see a pic in 250 nm because the absorbance is high compared to 260 nm, not à cause de contamination. For the profil of your RNA in bioanalyzer, it is normal because there is not ribosomal RNA in EV, so you cannot have a RIN number, you see juste à pic in the small RNA region (juste after the marker, between 20 and 200 nt).
I tried also to use RiboGreen, but the quantity is under the limite of detection.
So, you can juste use the bioanalyzer with picoKit to see the presence or not of your RNA and the estimated concentration pg/µl.
See the paper attached for more information.
good luck
Thank you all for your answers!
Ramia Safar I am relieved to hear that. However, when I compare my results of the RNA quality measurement with those from the paper, my RNA looks much worse. I measured my samples with the Bioanalyzer Nano Chip and the Tape station (high sensitivity RNA screen Tape). I have uploaded the results as pdf.
I would like to do a qPCR with my samples after the RNA isolation. Do you have experience with such small amounts of RNA?
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