We carried out time-course experiments with potato plants grown in pots with sand and fertilised with a nutrient solution. We harvested the plants after some weeks and frozen the shoots and the roots separately, in liquid Nitrogen, and stored at -80° C. Afterwards, we disrupted the roots with a Mixer Mill extracted RNA by a kit or with the trizol method. But the quality of the RNA seems to be not good. Do you have some explanation? Could it be because the growth conditions of the plants (stressing conditions)? Thank you in advance
Whatever the situation, if the plant is growing that means there must be some RNA in it. So, it is not supposed to be for stress condition. Probably it is for the handling of the sample. If possible try to isolate RNA from fresh plant parts. Collect fresh plant parts, add liquid nitrogen and immediately isolate RNA. Don't prefer to store at -80° C. Take all the precautions to avoid RNase. Treat all apparatus with DEPC before using them in RNA isolation. Clean the electrophoresis chamber with RNaseZAP and use fresh buffer for RNA.
Thank you for your reply. We also tried in parallel a RNA extraction of samples of another plant stored at -80° C and it worked. And I used always gloves, I don't know where could be the mistakes. Thank you for your tips!
Are you talking about a particular RNA of your interest? or the entire RNA?
I mean the entire RNA isolation, with the trizol protocol or with a kit that we have for this purpose
Dear Madam, as far as I know, In a growing plants the entire RNA can't get affected (degraded) due to an environmental stress.
So what I can guess from your explanation is that, there may be lots of endogenous RNase present in Potato plants which is causing the degradation during isolation. The endogenous RNase are generally inactivated by grinding materials under frozen condition which is followed by rapid disruption of cells in trizol reagent. The frozen tissue shouldn't get thaw during grinding.
Thank you for your reply, but I think that stress really can affect the quality of the RNA.
Here there is an example of a paper where it is said, that I have just found, searching for some tips after reading you:
Article Improved Protocol for Isolation of High-Quality Total RNA fr...
"Stress conditions leading to the accumulation of numerous secondary metabolites can affect RNA quality". The rest of the people in my lab makes RNA extraction as I made and they didn't have those problems of degradation, so I think it should be due to the stress conditions, more than to an inappropriate handling.
Many thanks again for your help!
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