I need to extract DNA/RNA from frozen diabetic rat whole brains.
To avoid RNA degradation I need to transfer the frozen tissue to the triazol mix as quick as possible before preceding with the experiment, so I can't use a scalpel to dissect out visible brain regions as I would need to wait for the tissue to thaw a little. I have been advised to use a pestle and mortar, and either:
- pulverise the tissue using pestle and mortar (with liquid nitrogen) and then taking a fraction of this powder
- crudely smash the tissue in liquid nitrogen with a pestle and roughly take the same region from each animal (for example the a part of the frontal lobe versus cerebellum which are both easy and obvious regions)
I have looked at other studies and all they state is that they used 'brain cortical homogenate' (from frozen tissue) - they do not explain what they did to obtain this region or whether they just homogenised the entire brain before aliquoting some of the tissue+triazol mix for RNA/DNA extraction.
Hi Elisabeth
If your brains are already frozen, I really don't see anyway that you can avoid having to thaw the samples briefly. With that said, you don't need to thaw rat brains very much before you can dissect them with a scalpel. How precisely you can do this is not something I would care to guess about but I think it would be more reproducible than smashing the brains with a mortar and pestle and then roughly identifying the cerebellum (or whatever region you are interested in). If it is not necessary for your experiment, I agree with Steven above that homogenizing the whole brain would be the wiser choice.
Ideally, you should dissect the brains immediately after extraction before freezing, I assume this is how people do it when they report using "brain cortical homogenate".
good luck
Jesper
Dear Elisabeth, if you have a cryotome at your university facility, you can try to cut the brains using it. Please, refer to the micropunching technique. For instance, here: http://www.scripps.edu/california/research/inia/methodology.html
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