RNA folklore?

[Peter Gegenheimer]

In <hrasal.223.000BAB94 at marc.cri.nz>, hrasal at marc.cri.nz writes:
>Greetings,
........
>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?

** It is, like all legends, based on a grain of fact.  The structure of RNase A 
is held together by disulfide bonds, so if it is autoclaved and then SLOWLY 
cooled, it may renature.  (This is why many RNA isolation protocols include high 
concentrations of BME.)  In practice, most RNA labs I know find that autoclaving 
gives buffers which are RNase-free.  Glassware should be oven-baked overnight 
for best effect, but autoclaving of dry (=fast cool) cycle works.  

>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.  

** Linear DNA is soluble in phenol at acid pH (<= pH 5). RNA and plasmid DNA are
not; they stay in the aqueous phase. This fact has been used to separate RNA and
DNA for at least 30 years. It has just been "rediscovered" as the basis of 
several commercial kits. The reason phenol should be equilibrated with the same 
buffer used for the aqueous phase is that liquid phenol is 88% phenol and 12% 
water, so when you do a phenol extraction, the aqueous component in the phenol 
mixes with your aqueous solution of nucleic acid. If you want close control over
buffer components and pH, pre-equilibration of the phenol is a must.

 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.
>Soon Lee 

** Other posters have good answers to this.  OH- mediated hydrolysis of RNA is 
dependent on [OH-] and temperature.  At 0-deg-C and pH 8.0, RNA is pretty 
stable.  At 100 degrees C, RNA will be hydrolyzed in unbuffered water.  Inclusion 
of EDTA (e.g., TE buffer) will chelate divalent cations which promote formation 
of OH radical and OH-.  

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