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Drosophila Developmental Northern

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue Jan 25 13:10:35 EST 2000


In article <26f4216e.2329ec1b at usw-ex0102-011.remarq.com>, martin
<martinNOmaSPAM at biologie.uni-freiburg.de.invalid> wrote:
> Hi everybody,
> even though my DIG-Northerns work pretty well, I have a problem
> with my
> loading standard. I use a DNA-DIG-rp49-probe, which (the literature
> says)
> is a constitutively expressed gene and has been used as a loading
> control
> for developmental northerns in other papers. I check my RNAs
> (total) in
> a
> photometer and am pretty accurate in respect to quantity while
> loading
> the
> sample on the gel.  The problem is, that rp49 always gives me
> totally
> different signal strengths after hybridization for the different
> developmental stages. Embryo and L1 is strong and the rest pretty
> weak.
> Unfortunately, especially those are weak that also do not show
> expression
> The RNAs do not seem to be degraded, since I see partially weak,
> but
> always
> distinct rp49 signals for all stages and no smear.
> What should I do? Is the photometer the reason? I do not look at
> the
> gel,
> since EtBr seems to make a pretty big background. Is Rp49 the
> reason.
> What other method could I use to quantitate the RNA?
> Thank you in advance!!
> Martin Struenkelnberg
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You could also check the loading (and transfer) by staining the blot
with methylene blue before probing.

IMO, that is the "best" way to check for equal loading of RNA.

Nick



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