Small riboprobes

Arne K Christensen via methods%40net.bio.net (by arnec from bio.umass.edu)
Fri Jan 22 14:50:25 EST 2010


Dear all,

I am in the process of designing 4 small riboprobes (20-23nt) that will 
be labeled with a fluorophore for use in ISH.  Two riboprobes are 
antisense and directed against VERY closely related isoforms, and the 
other two are their respective sense negative controls.  The reasons we 
have chosen very small riboprobes are:

1) because the genes are so closely related, there are only a few very 
small islands of divergence withing the mRNA sequences that we may target.
2) because we are not really set up for molecular work, it may be 
cheaper to order the labeled synthetic RNA sequences rather than 
transcribe our own.

The problem I am running into is that for any 20-25nt sequence I select, 
there is always a contiguous ~50%-70% of the sequence that is identical 
to other genes.  The selected sequences are so short, the chance of a 
redundant sequence anywhere in the genome of the species I am working 
with is very high.  To make things worse, I am working with a non-model 
species, and more than half of the transcriptome is not getting screened 
with a BLAST.

I would greatly appreciate any information people have regarding the 
selection of small ribroprobes for ISH.  Is 50% ID with other sequences 
divergent enough to get specific signal?

Thank you,
Arne



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