RE: In situ hybridization probes

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From:"Connolly, Brett" <> (by way of Marvin Hanna)
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You have encountered a common problem.  There are a couple of possibilities.
Some believe that sense transcripts are sometimes generated in the cell,
maybe as a regulatory mechanism. Or, your sense probe could be complementary
to another irrelevant gene transcript (or one not yet discovered) in those
particular cells. Do you BLAST the sequences against a database?
Or, your method is not stringent enough.

Things to do for starters:

1. decrease probe concentrations of you S and AS probes.
2. Increase hybridization temperature
3. Increase post-hybe wash stringency.
4. Use probes from a different sequence area of the gene; i.e. 3'UTR
5. Compete with unlabelled probes.

This all assumes that the root of the problem is in the hybridization.
Although DIG is not in mammalian cells may the anti-dig is sticking. Use the
Fab antibody and try decreasing the concentration.


Brett M. Connolly, Ph.D.
Merck Research Laboratories
Department of Pharmacology
PO Box 4
West Point, PA 19486
Ph. 215-652-2501
FAX 215-652-2075

> ----------
> From: 	Mike Bromley[]
> Sent: 	Thursday, January 20, 2000 6:32 AM
> To: 	'histonet'
> Subject: 	In situ hybridization probes
> Dear All,
> I have been staining formalin fixed paraffin embedded tissue sections
> for Epstein-Barr encoded small RNA's (EBER's) using the method of
> Helene Breitschopf from the Boehringer manual: Non-radioactive in situ
> hybridization  application manual 2nd edition.(published also in Acta
> Neurapathol (1992) 84:581-587).
> The method uses digoxigenin labelled RNA probes, the anti-sense is
> giving good strong staining in the right cells, unfortunately the
> sense probe is also staining the same cells albeit not as strongly.
> In the Boehringer manual p137, it states that hybridization with sense
> probes sometimes gives unexpected hybridization signals.
> Has anyone got any ideas about why the sense probe should apparently
> be specifically targetting the right cells?
> Mike Bromley
> Histology Department
> Paterson Institute
> Wilmslow Road
> Manchester
> M20 9BX
> UK

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