RNA folklore?

hrasal at marc.cri.nz hrasal at marc.cri.nz
Wed Dec 14 11:40:08 EST 1994


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 
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?

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 



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