Hello
I am working on a project where I need to extract RNA from my samples. For some reason, Nanodrop gives me values below [10 ng/uL] for almost all my samples, even though I saw a pellet in all of them, so expected to get at least [100ng/uL]. Does anyone know why the values could be so low?
Another question relates to protein extraction from the same samples. After I had added chloroform and extracted the RNA from my samples, I was aiming to extract protein as well, but the protein layer seemed to be almost non-differentiated from the DNA layer in the sample, so I didn't know what to do with it. I simply froze the samples in -80, because it was getting very late. Does anyone know whether I would be able to extract the protein if I thaw my samples and attempt again or are the samples now pretty much destroyed because of freezing them with residual chloroform and without any handling before?
Dear colleague:
I think you have problems with your RNA extraction. In my experience, when NanoDrop measurements are below 10 ng/uL, that measurements correspond to residual contamination. The pellet that you observed are just precipitated salts. Maybe you tissue sample is not enough, or your homogenization method is not the optimal for your tissue.
Regarding to your second question, we have performed the following protocol: Article Optimized solubilization of TRIzol-precipitated protein perm...
We have obtained very good results, but you must follow the protocol very closely.
I wish you the best!!!
Regards
Dear colleague:
I think you have problems with your RNA extraction. In my experience, when NanoDrop measurements are below 10 ng/uL, that measurements correspond to residual contamination. The pellet that you observed are just precipitated salts. Maybe you tissue sample is not enough, or your homogenization method is not the optimal for your tissue.
Regarding to your second question, we have performed the following protocol: Article Optimized solubilization of TRIzol-precipitated protein perm...
We have obtained very good results, but you must follow the protocol very closely.
I wish you the best!!!
Regards
Hello
Thank you for your answer, I have now re-precipitated the solution and hoping it will give me better values for Nanodrop.
Thanks for sharing the protocol, but I think that is for samples frozen in TRI. I had done that but then added chloroform to separate the RNA, protein and DNA layer. After that step, I eliminated the layer of RNA but did not do anything else to the samples, just froze them in -80. I'm wondering whether that will have an effect on the integrity of the samples (because there was probably some residual chloroform left and I'm not sure how that would affect the samples). Do you know if I can just follow the protocol as I would if my samples were still in TRI or whether there's additional steps I should follow?
Thank you
Hello Kaarin, how are you?
Well, RNA extraction with trizol you need to be very carefully with it, any error you lost your sample. As answered before, you are having trouble during RNA extraction. Small samples will yield lower quantity of RNA. However, with a big tissue sample, the volume of trizol will not be enough to disrupt the tissue and release RNA. So, you will also get lower quantity of RNA. In addition, try to use a small volume of water like 10ul to resuspend your RNA pellet and then measure in the nanodrop. If the RNA concentration is too high you can dilute a little bit.
Regarding to protein extraction, I never performed in my samples. I think the chloroform is not the problem. The supernatant is actually saline buffer with the RNA, the phenol-chloroform is used to precipitate the proteins. We use a similar protocol in DNA extraction in samples with high quantities of proteins. If you follow the Trizol instructions for protein isolation I think you will not have any problems. Good luck.
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