The rats will be subjected to chronic alcohol vs controls. The brains will be collected, RNA extracted and the levels of candidate genes will be analyzed by RT PCR.
Which areas of the brain are most promising to see the meaningful differences.
Factors to consider:
Some important areas don't contain cell nuclei. Some important areas are small and hard to locate and collect. Shall we first separate brain areas and then freeze? Or freeze, then cut and cut out frozen areas?
So the question is: among the areas related to chronic alcoholism, which ones are easy to locate and collect?
I think cerebellum, striatum,, hippocampus and prefrontal cortex, but my knowledge of brain areas and of alcohol research is limited. Please help!
Hi Max,
The brain areas you have indicated in your list should do the job well. I would add amigdala, ventral pallidus with accumbence and septum. These limbic structures are likely to be affected most, although in general terms any brain structure should get troubled in one way or other.
As regards to the tissue extraction, typically this is done immediately after extracting the brain on ice. Cutting areas of frozen brain is not a good idea and is unlikely to work, so I would suggest you to take the first option.
Best luck,
SV
Hi Max!
Ron Paletsky used to kill mice fast and freeze brain immediately in some specific liquid to avoid degradation. How he extracted specif tissue I do not know. Then I, guess, you can do cryosectioning following by dissection. After sectioning you fix in ethanol and dry you samples. This allows you to avoid RNA degradation in the process of dissection. Do you have dissecting scope? Laser capture microdissection would be the best, let me know if you will use this option, I can help.
It would be interesting to know opinions of people who are experts in brains.
Best!
Julian Rozenberg
actually for PCR perhaps extraction of the frozen tissue should also work as you do not need much.
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