Heat stability of RNA
Rimantas.Plaipa at gf.vu.lt
Tue Oct 19 04:07:58 EST 1999
John Scott Meschke wrote:
> 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?
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.
Department of Biochemistry and Biophysics,
Ciurlionio 21/27, Vilnius 2009, Lithuania.
E-mail: rp010gf at voruta.vu.lt
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