RP49 in RNAse Protection Assays

[Lauryl Nutter]

Hello Fellow Drosophilists!

I am currently doing some RNAse Protection Analyses (RPAs) on total 
Drosophila RNA isolated from either whole adult flies, adult heads, 
ovaries or testes.  I am using RP49 as a control probe for target RNA 
quality and quantity.  I have been using the A260 for determining RNA 
quantity of each sample.  I consistently find that with the RP49 probe, 
the signal is much lower (approx. 5 to 10-fold, although I have not done 
a densitometric scan) with the testes RNA, while all the other samples 
(quantitated in series on the spec with the testis RNA) show equal 
signals.  So my question is, has anyone else out there found this type 
of result, ie. that RP49 RNA is in lower abundance in testes.  I know 
that it is supposed to be equally expressed everywhere, I'm just 
wondering if anyone has actually done the experiments on testis 
expression.

One suggestion that I have from a colleague here (who works with 
Xenopus, not Drosophila) is to run an ethidium-stained formaldehyde gel 
with my target RNA and see if the ribosomal RNA bands are equally 
intense.  This sounds like a good suggestion to me and I'm going to be 
trying it this weekend...

In the meantime, if anyone else has any other suggestions, I'd be happy 
to hear them.

Thanks for your help.

Lauryl Nutter
Dept. Biological Sciences
The University of Calgary
lmjnutte at acs.ucalgary.ca