Laser Capture Microdissection

From:Margaret Kelly

I am using the Arcturus Pixcell II System for LCM.  I am extracting RNA 
from microdissected lung, and ultimately want to examine quantitative gene 
expression. I have found the system fairly easy to use, but would recommend 
attending one of the courses run by Arcturus at their headquarters in 
Mountain View, California. I attended their level 2 training course which I 
found very useful even though I was already using the method. I picked up 
several practical hints and found the course extremely well run and 
worthwhile. I haven't been able to obtain enough RNA from a small enough 
number of cells, and am now evaluating linear amplification of RNA which 
hopefully will solve my problems. Arcturus makes such a kit, "Riboamp" as 
does Ambion, "Messageamp aRNA", which we are comparing. As others have 
mentioned, you need to work fast, and not work on too many slides at once. 
For RNA work, the tissue needs to be frozen in OCT and not fixed in 
formalin. I cut the sections and then freeze them at -70 C until needed. I 
only take out 2-3 at one time as the microdissection takes time, and the 
tissue should be captured as soon as possible, at least within 2 hours of 
removal from freezer. Arcturus has good protocols on their website. Be sure 
to put the slides through the full cycle of hydration and dehydration to 
xylene because the OCT is only removed during the water wash. If the OCT 
isn't removed, you will just get a great 'imprint' of the cells on the cap 
but the tissue won't be removed from the slide.

Looking forward to hearing about other peoples experiences with this technology


Margaret Kelly
Centre for Gene Therapeutics
Dept. Pathology and Molecular Immunology
McMaster University
Hamilton, ON

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