ISH control

David Micklem dmicklem at
Fri Jun 19 02:19:09 EST 1998

In article <Eus28w.2B4 at>, rjv at (Richard
Vernell) wrote:

>My question is:
>What is the best control for in situ hybridization with RNA probes.  Is an
>irrelavent probe as valid as the sense probe?  I will be hybridizing many
>different probes and wondered if i could use one control such as the multiple
>cloning region of pBluescript.  This should control for nonspecific
>binding of RNA to the tissue sections, espececially if i also include
>pretreatment with RNase as a control .  I do not see the reason for
>making sense probes for each cDNA.  Please enlighten me... especially with
>respect to the opinions of reviewers on this matter.  Thank you.
>Rich Vernell

Hi Rich,

I agree with you on this, though I don't know what reviewers think. 
Perhaps try to pick a negative control probe with at least a similar base
composition to your real probes? I never did see why the sense probe should
be considered a good control. Its even (usually) made with a different
enzyme so could have considerably different yield and incorporation of
whatever modified nucleotide you are using...

Personally, I'd also keep RNAse as far away from my samples as humanly
possible, so I'd ditch that particular control.


D.R. Micklem,                            Time flies like an arrow... 
Beckman Institute,                   Fruit flies like a banana.       
Stanford, Ca 94305 USA           Email:dmicklem at
Unsolicited mail will incur a US$100 processing charge.

More information about the Methods mailing list