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:
>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 
>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 


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

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