High background in FISH with TSA

Anant Patkar apatkar at biosci.cbs.umn.edu
Sat Mar 13 13:22:39 EST 1999



Stefan Burde wrote:

> Enrique,
>
> If your mRNA is fairly abundant, you can probably get away with directly
> labeled fluorescent probes.  They will give less signal per probe
> molecule, but will also have much lower background.  If you need some
> signal amplification, and endogenous biotin is likely to be present,
> then digoxigenin should indeed work better.
> Hope this helps,
> Stefan
>
> Stefan Burde
> Los Alamos National Laboratory
> Los Alamos, NM, EE.UU.
> burde at lanl.gov

We are also starting some work on identifying some sequences at the mRNA and
possibly
at the DNA level.  Can anyone tell me the sensitivity of FISH with use of
directly labeled
fluorescent probes?  Right now I am just thinking about FITC-labeled UTP and
PCR to
generate the probes.  We have plans on using this technique along with flow
cytometry
at some point.  You mention that digoxigenin labeled probes should work
better in terms
of sensitivity. What is the best people have been able to do so far?  I have
seen some
references for 100 copies of mRNA, but nothing less.  How about single
copies of a DNA
sequence?  While we are on this topic, has anyone had better luck with the
Alexa-488 modified
UTPs from Molecular Probes?

Thanks a lot from a FISH beginner.

Anant Patkar
(apatkar at biosci.cbs.umn.edu)
University of Minnesota




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