Drosophila actin probe for RNase protection wanted

Richard M. Rohan rrohan at umabnet.ab.umd.edu
Wed Mar 8 18:13:39 EST 1995


On 7 Mar 1995, Shahram Mori wrote:

> Bernard Khoo (WCI) (bcek1 at mole.bio.cam.ac.uk) wrote:
> 
> : I wish to use an actin probe to normalise RNA levels in an RNase protection
> : assay. I have a mouse probe, but I'm not quite sure if this is homologous
> : enough to be suitable.
> 
> I would run a Gen-bank search for both and align them. If they are highly
> homologous ( which I am pretty sure they are) you can use it. This will save
> you money ordering a probe.
> 
I am not sure that there will be enough homology for this to work.  Ambion
has a mouse actin riboprobe that differs from rat in 10 of 250 nucleotides.
The divergence from Drosophila must be much greater.  RNase conditions in the
protection assay can supposedly be adjusted to decrease the cleavage at 
single base mismatches.  However, I have been told that two adjacent mis-
matches are fatal.  We have successfully made species specific probes for
RPA by RT-PCR.  Find short regions of homology in actin sequences and use
oligos to these regions in PCR of reverse transcribed Drosophila RNA.
Alternatively, you might try Ambion's 18S riboprobe.  It protects rRNA from
many mammalian species.  Maybe it will work for Drosophila.  We use it	
routinely to normalize RPAs of RNA from rat tissues.
Rich Rohan
University of Maryland





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