RNA dot blot protocol?

Teresa Bori-Sanz via methods%40net.bio.net (by mtbori At pcb.ub.es)
Thu Dec 28 11:52:46 EST 2006

Hi Dr. Klaus Eimert, 
I was wondering if somebody got back to you. I am also trying to do a DOT
BLOT but I would like to try it to detect tRNA. If so, could you let me know
what was the best protocol?
Best wishes, 
Hi evreybody,
I am looking for a fast and "fool-proof" RNA dot blot protocol.
I want to do some ISH on paraffin sections using DIG-labeled probes. To 
make sure that my probes are OK and specific, I thought to do a RNA dot 
blot for control. 
I thought to just spot the total RNA on the membrane and let it dry (no 
manifold or such). The protocols I found in the books and on the web differ 
quite a bit, though. In some, RNA is spotted on the dry membrane, some 
prewet with water or SSC (2x to 20x). Also, some don`t denature the RNA at 
all, some "boil" (65-70°C) and some use NaOH.
So, does anybody care to share her/his good/bad experience with a certain 
protocol? Any traps I should to avoid?
Thanks in advance!


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