carrier/blocking nucleic acids for ISH

Peter Ellis via methods%40net.bio.net (by pjie2 from cam.ac.uk)
Thu Jan 14 07:15:00 EST 2010


On Thu, 14 Jan 2010, Dunowska, Magda wrote:
>
> Hi everybody, I have a quick question related to carrier/blocking 
> nucleic acid used as part of hybridization buffer for ISH. Some 
> protocols use salmon sperm DNA, some tRNA and some both. We are trying 
> to detect an RNA virus with a DNA DIG labelled probe. Is there any 
> advantage to use one versus another versus both?

It depends what sequences are present in your immobilised target and in 
your labelled probe.

Both salmon sperm DNA and tRNA will block any nonspecific nucleic acid 
binding to the slide / membrane surface.  Salmon sperm DNA will also block 
highly repetitive sequence within the probe and target, such as 
dinucleotide and trinucleotide repeats.  Cot-1 DNA is also often used: 
this will block other repetitive sequences such as LINEs and SINEs, 
although by and large the blocking will be species-specific.  Human Cot-1 
DNA will block human repeat sequences, etc.

I presume you are trying to detect viral infection, so your immobilised 
target will be a cell lysate containing DNA/RNA from your species of 
interest, and potentially the viral RNA depending on infection status.

If there is any possibility that your viral sequence will cross-react with 
endogenous retroviruses, I would suggest you block with the appropriate 
Cot-1 DNA, plus some tRNA to block the slide/membrane surface.  If 
not, then tRNA alone may be sufficient, or tRNA + salmon sperm DNA if 
you're getting non-specific smears.

Peter


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