>I am looking for advice in non-radioactive detection of very low
>abundance mRNA species in mouse brain. Standard biotin or dig-labeled
>protocols do not work, and for logistical reasons we do not want to use
>isotopes (although P-33 does detect these mRNAs). I am thinking of trying
>either a tyramide amplification strategy, or perhaps an enzyme-linked
>flourescent approach. Or maybe both together. Any help in this area would
>be Much appreciated!

Another alternative would be plain old enzymatic detection - both alk
phos. and HRP can be used and will give quite a bit of amplification. I've
also used tyramide, but it can be fussy to start dealing with; most labs
will already have experience with AP and/or HRP, so using those might
be easier.

However, have you considered that the reason you aren't getting signal is
due to your probe or fixation or any of the other zillion thiings that
thwart mRNA ISH and not the detection?

Good luck!

Tamara


|--------------------------------------------------|
 Tamara Howard
 Department of Cell Biology and Physiology
 University of New Mexico - Health Sciences Center
 Albuquerque, NM 87131
 thoward@unm.edu
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