RNase Protection Assay; non-specific?

Rolands G. Aravindan aravind at wsunix.wsu.edu
Thu Feb 26 02:46:25 EST 1998


Thanks David for your suggestions and questions. In order to answer your
questions, I had to meet Professor Sarkar to get to see the blot again.
Though I could not actually see it again, I was told that by contacting
the postdoc who had actually done the work, the mystery of the additional
band could be sorted out. It apparently turned out that the extra band was
generated artifactually by the Cyclophilin standard, though I could not
quite understand how, nor was it well explained to me (I have not done RPA
before either). I have tried to explain your questions below:

On 22 Feb 1998, David L. Haviland, Ph.D. wrote:
 
> 1)  You make no mention of the "ever-needed" tRNA control lane.  This is
> almost always shown and should be absolutely blank.  There should be no
> hybridzing signal and theoretically no signal from the probe either.

I could not see the blot. I do not remember seeing an empty, clean lane,
though.
 
> 2)  What is the purpose of the RPA?  5'-end detection?  Overall message
> detection?  In any case, the signal should be visibly less (base pair wise)
> than that of the probe alone.  I usually design my probes to be 200-250 bp

Overall message detection. yes the 'nonspecific' was visibly lesser, by
about 100 bp - infact, this was the reason that the referee's question was
kind of puzzling to me!

> 3)  Can the additional signal possibly be due to the probe folding on
> itself?  GCG -RNA fold might lend some information. 

The explanation given to me did not mention the probe folding on itself.
It had something to do with the inhouse gene mRNA (cyclophilin)
hybridization. Sorry I could not say it any better.
 
> 4)  Is the sample RNA poly A+ message or total?

Total, actually.
   
> I understand that it is a dose response increase of your message that
> you're looking for.   However, would primer extension be of use?  Normally,
> these two methods are played "off each other" as a dual evidence for
> suggesting that transcription starts at a particular nucleotide, given how
> RNA folding can render a false signal.  If your RPA is based on the very
> 5'end of your message, this might be something to consider.

Sorry, David, I don't quite understand. How does primer extension fit in?
It is kind of greek to me at this point. Can you point me towards what it
means, towards some basic fundamental references, tech notes? Also, how
can RPA be based on the "very 5' end of the message"? Isn't it based
rather on the hybridization of a big antisense sequence (in this case,
POMC RNA, 250 base probe)? I apologize for my ignorance in these aspects.

> Hope this helps

Sure helped me to understand better and learn. Have a long way to go,
though.

I have another question. Do I have to prepare all reagents for RPA in DEPC
water as a general rule? I am supposed to try and set up RPA back again in
the lab and from what I could gather this is what I have to do. I do not
want to end up finding that there are certain reagents that need to be
without DEPC.

Thanks 

aravindan
aravind at wsu.edu





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