Heat stability of RNA

Rimas Rimantas.Plaipa at gf.vu.lt
Tue Oct 19 04:07:58 EST 1999



John Scott Meschke wrote:
> 
> List-
>         This may seem like a silly question.  What is the probability
> that RNA be damaged by exposure to 95C for 5 min?  I realize that for
> heat release RT-PCR of viral RNA this is routine to open the viral
> capsid.  However, when doing an RNA extraction prior to RT my gut
> reaction is to still heat the RNA before addition of the reverse
> transcriptase in order to reduce secondary structure.  Is this heating
> potentially problematic?
> 
> Curious,
> 
> Scott

I use 95 C for several min in the buffer with 1 mM EDTA for RNAs of ~
100 nt, and they survive this treatment perfectly (according to PAGE).

The rate constant of the hydrolysis of the single phosphodiester bond in
RNA at 95 C and pH of ~7 is about 10^(-7) 1/s .(My reprints on this
subject are at home, write me personaly if you are interested in the
exact numbers and references). So the probability to be damaged in 5 min
(300 s) for one bond in RNA is aprox 0.003 %, for the whole molecule of
100 nt long RNA - 0.3%, 1000 nt - aprox 3 %. But these are the minimal
estimations at pH where the rate of hydrolysis is at minimum and without
divalent ions (Mg). If your pH is higher than 7 and/or the solution
contains Mg the rates may be 100s of times higher. 

-- 
Rimantas Plaipa,

Department of Biochemistry and Biophysics,
Vilnius University,
Ciurlionio 21/27, Vilnius 2009, Lithuania.

E-mail: rp010gf at voruta.vu.lt
Phone: (370-2)-650381 
Fax: (370-2)-235049
---



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