Hello everyone,
Being naive to the process of RNA isolation from mouse kidney, I wanted your help in adapting an optimal protocol for the following:
1. Preserving mice tissue for RNA. For e.g. Flash freezing in liq. N2, using RNAlater or similar techniques.
2. Isolating RNA from tissue preserved with any of the aforementioned methods.
I would be delighted to have your responses.
Best,
Sudip
I personally do not like using RNA Later because it interfered with procedures for our downstream application. I think RNA Later contains high amount of salt (please do not quote me on this one) that needs to be removed before you isolate RNA in order to avoid issues with whatever you do afterwards. I place tissue in RNA isolation solution (TRIzol, RNAzol, etc), flash freeze the sample in liquid nitrogen, transport in it or on dry ice to the lab, and store them until RNA isolation because of the volume of the tissue we process in my lab. The key to isolating RNA from tissue samples, I am sure others would agree, is to get your samples homogenized in the isolation solution so that RNase will not degrade RNA.
I personally do not like using RNA Later because it interfered with procedures for our downstream application. I think RNA Later contains high amount of salt (please do not quote me on this one) that needs to be removed before you isolate RNA in order to avoid issues with whatever you do afterwards. I place tissue in RNA isolation solution (TRIzol, RNAzol, etc), flash freeze the sample in liquid nitrogen, transport in it or on dry ice to the lab, and store them until RNA isolation because of the volume of the tissue we process in my lab. The key to isolating RNA from tissue samples, I am sure others would agree, is to get your samples homogenized in the isolation solution so that RNase will not degrade RNA.
Hi Sudip. My procedure has been to keep at 4oC for 24 hours in at least 10 volumes of RNAlater, store long term at -20oC or colder, thaw, then bead beat in in Trizol.
Yasushiro's procedure also looks good, as long as you start homogenization when in frozen state. The critical element is having the RNA in the thawed tissue protected from degradation.
Best regards
Roger Latham
I agree with both. Snap freezing your tissue vin extraction buffer would help.
All the best.
Shefali
Hi guys,
Do you think I can use the same protocol described by Yasuhiro for tumor tissue? If I want to freeze the samples for a long period before to perform the extraction, should I homogenize the tumor tissue with Trizol before to freeze? In this case, should I use the volume described in the trizol datasheet (1 ml for each 50-100 mg of tissue sample)?
Cheers,
Adny
Adny,
Tissue protocol is same for all provided your tissue is fresh and not in a paraffin block.. You can use trizol detasheet for the extraction.
Best
Shefali.
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