monitoring RNA synthesis

William Sun william at neuro.usc.edu
Wed Sep 4 14:10:54 EST 1996


Hello all,

I am having some problems monitoring RNA synthesis in vivo.  I wish to test
the efficacy of actinomycin D in blocking RNA synthesis in rat brain.  I 
implant cannulas into both sides of the brain, infuse actin. D. into one side
and saline control into the other side.  After 1 hr I infuse 3H-uridine into
both sides of the brain.  I remove the brain after 1 more hr, extract RNA by
homogenization in Trizol.  I spot the RNA on filter paper in duplicate, one
unwashed and the other washed with cold 10% TCA.  I then count by scintillation
counting, computing the ratio between washed and unwashed filters. I expect 
the side infused with actin. D. to have a lower ratio of counts than the one
infused with saline.  However, I don't see this result consistently.  Half of
the rat brain I tested show lower ratio of counts on the actin. D side, but
the other half show higher ratio!  I am not sure what I could modify in my
experimental protocol, if you see any potential flaws in the design or an
alternative method, please reply.  Thanks for any suggestions.

-William


-- 
William Sun, Ph.D.                     Phone: (213)740-3406
Neuroscience Program                   FAX:   (213)740-5687
University of Southern California      Pager: (310)243-9878 
Los Angeles, CA 90089-2520             http://rana.usc.edu:8376/~wisun/



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