Worm Breeder's Gazette 11(1): 68

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In situ Detection of mRNAs with Digoxigenin-Labelled Probes

Leon Avery

Figure 1

Hoping they might eventually prove more sensitive than biotin-
labelled probes and allow better localization than [35S]-labelled 
probes, I have been doing in situ hybridizations with digoxigenin-
labelled probes.  When I stained pure DNA or RNA fixed to slides, I 
saw punctate staining.  The number but not the brightness of the 
points depended on concentration, suggesting each point was an 
individual molecule.  (A molecule would have contained 1300 
nucleotides of labelled DNA carrying 60 molecules of digoxigenin.  
From the number of points, each contained at most ten times that.) him-
8 squashes stained with labelled vit-2 DNA (made from the plasmid DJ1-
B given me by Peg MacMorris) by the protocol below had punctate 
staining in adult hermaphrodite intestines, but not in other tissues 
or adult male intestines.  There was no staining when a non-homologous 
probe was used.  The only exception was a small amount of punctate 
staining on the surfaces of gonads with both vit-2 and nonhomologous 
probes.  mab-3 mutations are known to cause yolk expression in males, 
and both male and hermaphrodite mab-3; es 
stained.  The brightness of the points was the same as I saw with pure 
DNA or RNA, suggesting they might be individual mRNA molecules.  If so,
I detected <1% of the message: there were <100 points in an 
intestinal cell, where a rough calculation suggests there should be 
10+E4-10+E5 molecules of vit-2 mRNA.  I hope this dismal detection 
efficiency will be improved by optimizing probe, fixation, etc.
[See Figure 1]
The acetylation step reduces non-specific probe binding, and 
powdered milk reduces non-specific antibody binding.  The 
hybridization conditions were worked out using pure RNA fixed to 
slides.  Aside from these, it should not be assumed that there is a 
good reason for any step.  Contact me for a current, excruciatingly 
detailed protocol.

Figure 1