RNA folklore?

Curt Ashendel ashendel at aclcb.purdue.edu
Wed Dec 14 17:24:01 EST 1994


On Wed, 14 Dec 1994 11:40:08, 
hrasal at marc.cri.nz  <hrasal at marc.cri.nz> wrote:

>There are two things that  I'm curious about.  When I first started 
>working with RNA, I was told that autoclaving inactivated RNAses, but the 
>RNAse protein will (after a certain period, 24 hours or so), reform into the 
>active enzyme.  I had assumed that this was stated in Stryers Biochemistry.  I 
>was wrong.  Actually, it was denaturation of ribonuclease in 6M guanidine HCl 
>followed by removal of the guanidine by dialysis.  So, is reactivation of 
>RNAse activity after autoclaving an urban legend, or is it real?

I believe that RNaseA is stable to autoclaving temps in nondenaturing 
neutal solutions. I would not rely on it to kill RNases in general (which 
may or may not be as pancreatic RNaseA).  It is done usually to keep all 
solutions free of microbes, which naturally produce and secrete RNase.

>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.

pH 8.0 is not sufficiently alkaline to seriously damage RNA in the cold, 
but most people use pH 7.0 TE or DEPC'd water for making RNA solutions and 
also most use acid (pH 4.0) buffer with non-pH-equilibrated phenol for the 
"Acid phenol" RNA prep method, since DNA (only) is relatively selectively 
lost to the interface/organic extraction phase.

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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