Help with ISH using labeled oligos

Steve Marshall steve at genedetect.com
Mon Sep 9 01:33:26 EST 2002


Hi Oswaldo

Firstly, since you detect rRNA (a highly abundant RNA species) you are
able to get away with end-labeling your oligos. End labeling (as
opposed to tailing) adds one DIG molecule to the 3' end of the oligo
probe. Tailing on the other hand usually adds up to FIVE labels
incorporated into a tail of up to FIFTY additional nucleotides (so the
labeled nucleotides are usually spaced every five nucleotides in the
tail). The tail is flexible and may lead to some increased background
compared to an end-labeled oligo, yet it will be far more superior in
terms of sensitivity of the oligo, since each oligo molecule now has
five labels (i.e. DIG molecules) attached to it.

If you only require single labeled probes (i.e. where your target is
abundant, i.e rRNA, on a northern or dot blot) it doesn't matter
whether the DIG (or biotin or FITC) molecule is located on the 3' end
(as can be added by end labelling) or on the 5' end (as can be added
enzymatically, or more commonly during probe synthesis).

If however you are performing normal, tissue in situ hybridization for
a particular mRNA species, you should use either a tailed probe (5
labels per probe) or a proprietary labeled probe (like the GreenStar
probes we make that incorporate ten labels per probe).

In terms of your specific questions then:

1. I was thinking about labeling the probe with biotin 
> and then using an avidin-biotin-AP complex for signal amplification and 
> detection. However, I have got confused with the different options of 
> labeling with biotin, this is, is it better to label the 3' end, or the 
> 5' end, or both?. 

Ans. If you only require that the probe be labeled with a single
biotin molecule it doesn't matter whether the biotin is on the 5' or
3' end. The choice is decided for you however, since most oligo
suppliers only pre-label their oligos on the 5' end. So go with that.

2. How will that affect the stability of the hybrids?.

Ans. It won't affect the stability at all. It was in the old days
common to incorporate labeled nucleotides within the oligo....but that
did seem to alter the stability of the hybrids and is now not
recommended.

3. Also, I have read lately about the possibility of labeling the
oligo
> directly with Alkaline Phosphatase. This would be nice because the 
> AP-conjugated avidin steps would be skipped, but wouldn't that 
> compromise sensitivity because we are not going through the signal 
> amplification step that the avidin-biotin-AP provides?. 

Ans. Exactly. The suggestion if this is your preferred option might be
to use a cocktail of non-overlapping probes labeled with AP.

4. Most protocols 
> using this new direct AP incorpotration to the probes are intended for 
> membrane-based hybridizations and they use chemioluminiscence for signal 
> detection claiming equal or higher sensitivities than more traditional 
> antibody-based protocols, but I have not found references on the 
> suitability of these systems for ISH using conventional AP sustrates 
> like NBT/BCIP.

Ans. Neither have I suggesting they may be quite insensitive where
target is scarce, although if they are applicable to any form of in
situ hybridization, ISH of rRNA may be the one case, especially if you
try a cocktail of AP labeled probes.

5. which probe label 
> would be better for my application, Biotin or AP and if it is biotin, 
> which labeling option is more adequate, 5' or 3'. 

Ans. I think the biotin option is better, although you will need to
watch out for background staining (due to high endogenous biotin
levels) in two particular tissues, liver and kidney. In fact we
suggest to our customers they NOT use biotin labeled oligos if they
are investigating these tissues. Im not sure if you are or are not?
Why don't you just stay with DIG? DIG usually gives quite low
background and you already have it working? We can supply DIG
pre-labeled probes. Regards 5' versus 3' labeling. There is really no
difference.

Regards
Steve

www.genedetect.com
"World's largest selection of gene probes"


oswaldo <oswaldo at REMOVETHISiats.csic.es> wrote in message news:<3D786382.3080107 at REMOVETHISiats.csic.es>...
> Hi,
> 
> I am designing experiments involving a non-radiactive ISH protocol for 
> the detection of pathogen-specific rDNA sequences with short DNA oligos 
> (22-25 nt) in animal tissues. I have previously carried out similar 
> procedures using oligos to which I attached a  DIG molecule using the 
> Boehringer (nowadays roche) 3'-end oligo labeling kit, and I usually got 
> good results because the sequence to detect is highly specific and very 
> abundant in infected tissues. The only pain is the hands-on time of the 
> procedure.
> 
> However, now I am thinking about ordering pre-labeled oligos from a 
> supplier. The trouble comes with the choice of labels. So many labels, 
> so little time... I was thinking about labeling the probe with biotin 
> and then using an avidin-biotin-AP complex for signal amplification and 
> detection. However, I have got confused with the different options of 
> labeling with biotin, this is, is it better to label the 3' end, or the 
> 5' end, or both?. How will that affect the stability of the hybrids?.
> 
> Also, I have read lately about the possibility of labeling the oligo 
> directly with Alkaline Phosphatase. This would be nice because the 
> AP-conjugated avidin steps would be skipped, but wouldn't that 
> compromise sensitivity because we are not going through the signal 
> amplification step that the avidin-biotin-AP provides?. Most protocols 
> using this new direct AP incorpotration to the probes are intended for 
> membrane-based hybridizations and they use chemioluminiscence for signal 
> detection claiming equal or higher sensitivities than more traditional 
> antibody-based protocols, but I have not found references on the 
> suitability of these systems for ISH using conventional AP sustrates 
> like NBT/BCIP.
> 
> I would appreciate if anybody out there has some experience with related 
> ISH protocols and can give me some tips on deciding which probe label 
> would be better for my application, Biotin or AP and if it is biotin, 
> which labeling option is more adequate, 5' or 3'. Thank you in advance,
> 
> Oswaldo



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