RNA isolation following apoptotic cell labeling - (Feb/25/2009 )

I am trying to find a way to identify apoptotic cells in a frozen tissue section prior to laser microdissection of NON-apoptotic cells and either protein or RNA isolation. TUNEL wont work as it is destructive to proteins and RNA.

Does anyone have any ideas?

Thanks in advance.

tmbonney on Feb 25 2009, 04:05 PM said:

Does it have to be done in tissue sections? If you can dissociate the tissue, you could run it through FACS and then do RNA iso from the selected population. I know FACS can be used to identify apoptotic and non-apoptotic cells, but I'm not sure how. Try the Flow Cytometry forum if this is an option.

In many cell types, you can identify apoptotic cells visually. You'll see more "vesicle"-looking bubbles in dying cells. It's particularly easy if a decent percentage of your cell population is apoptotic. If your tissue is transparent and you have a good light microscope, this should work just fine.

Apoptotic cells should also have condensed nuclei. This can be visualized using DAPI or Hoechst. Acridine orange can also be used. Acridine orange stains red when bound to RNA, but green when it binds to DNA. However, it can not enter a normal nuclei. Dying cells (or mitotic cells, but you should be able to tell the difference) will have permeablized nuclei, and the acridine will be able to get in and these cells will fluoresce green while non-mitotic viable cells will not.

-Carlton

tmbonney on Feb 25 2009, 02:05 PM said:

Hi all, I am a new member of forum. Would a newcomer be warmly welcome here? Good day you guys!!!
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