Iım trying real time RT-pcr with samples from brain sections. I have
injected a virus into the striatum of a rat which among other things
expresses GFP. Iıd like to collect fresh brain, and cut a rough 5-mm slice
around the injection site (around where the needle went in). Ideally Iıd
freeze this on dry ice to keep the mRNA safe, and at a later time point cut
large sections (200-300 microns) using an AO 860 sliding microtome. I can
drop these immediately in buffer, visualize the GFP under the microscope and
roughly dissect out the transduced region under the scope. I could hand
dissect the tissue, but it will dilute the effect of the virus since the
entire region is not affected.
So, my question is, how reasonable is this approach? In particular, can
does the initial dry ice freezing-thawing during section and dissection hurt
the mRNA? How quick do I have to be to prevent loss? Is there a
particularly good buffer to prevent Rnase activity (autoclaved PBS?)?
Perhaps most importantly, will I be able to cut thick sections from an
unfixed brain on the sliding microtome? Thin sections from unfixed brain
tend to fall apart.
I know this isnıt the ideal system, but Iım trying to work with the
equipment I have. An alternative would be to take a quick section, look
under the scope and then do a rough dissection of the frozen mounted brain
once I start to see a signal.
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