RNA folklore?

NICHOLAS THEODORAKIS ntheo at welchlink.welch.jhu.edu
Wed Dec 14 12:28:37 EST 1994


In article <hrasal.223.000BAB94 at marc.cri.nz>,  <hrasal at marc.cri.nz> wrote:
>Greetings,
>
>I currently do a lot of work with RNA (specifically, fruit RNA).  As with work 
>of this nature, I take all the usual precautions and have had no problems.  
>Recently, I've been thinking about all the precautions that I take.

>There are two things that  I'm curious about.  When I first started 
<snip>
>Also, phenol is used in the RNA extraction protocol that I use.  The phenol is 
>equilibrated with tris-HCl as prescribed by Sambrook, Fritsch and Maniatis' 
>Molecular Cloning.  In it, they state that the phenol should be equilibrated 
>to pH >7.8 as DNA (and I assume RNA) partitions into the organic phase at acid 
>pH.   Now, RNA will hydrolyse under alkaline conditions.  My second question 
>is this: At what pH and/or concentration of alkali does RNA hydrolyse?  I've 
>checked Sambrook, Stryer and the Merck index, unsuccessfully.  FYI, I use 
>phenol equilibrated to pH 8.0.  For Northerns, I blot my RNA onto HybondN+ 
>with 50 mM NaOH.
>
>Comments, references etc welcome.
>
>Soon Lee 

Actually, it is possible to differentially extract RNA (vs DNA) into the 
aqueous phase during a phenol extraction, which I believe is the basis 
for the Chomzinski (probably not even close on the spelling) -type 
methods of *acidic* phenol extraction (as in the popular Trizol reagent).

As for the alkaline hydrolysis, I would think rather than there being a 
discrete point at which the RNA hydrolyzes, that the problem gets worse 
with increasing pH, time, and temperature.  For reference, I once did a 
"reverse" dot-blot by labelling the RNA with kinase after partial 
hydrolysis with Tris, pH 9, for 10 min @ 90 C.

I believe that the alkaline transfer during blotting does partially degrade 
the RNA, but this is done on purpose to facilitate the transfer of large 
RNAs.

					Nick


-- 
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
			Nick Theodorakis
			ntheo at welchlink.welch.jhu.edu
			Johns Hopkins Medical School, Baltimore, MD



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