Does heating influence RNA quality? My understanding was the RNA should not be heated and kept on ice.
Trizol protocol: http://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf
Thanks,
Rami
Hi Rami,
The short heating step (55-60 degrees C for 10-15 minutes) at the end of the protocol is used to promote the resolubilisation of the RNA.
After the RNA has been precipitated, it can sometimes be hard to get it to go back into solution again; the heating to 55-60 degrees helps with this. While it's true you don't want to heat RNA (or even store it at room temperature for any length of time), the short incubation time here (10-15 mins) will allow fairly minimal degradation of the RNA while increasing the total amount of useable RNA - i.e. RNA in solution, not inacessible in a pellet - at the end of the protocol.
I hope that makes sense and answers your question - please let me know if not.
Cheers,
Dan
Hi Rami,
The short heating step (55-60 degrees C for 10-15 minutes) at the end of the protocol is used to promote the resolubilisation of the RNA.
After the RNA has been precipitated, it can sometimes be hard to get it to go back into solution again; the heating to 55-60 degrees helps with this. While it's true you don't want to heat RNA (or even store it at room temperature for any length of time), the short incubation time here (10-15 mins) will allow fairly minimal degradation of the RNA while increasing the total amount of useable RNA - i.e. RNA in solution, not inacessible in a pellet - at the end of the protocol.
I hope that makes sense and answers your question - please let me know if not.
Cheers,
Dan
Daniel Dehany Scott
Thank you for your answer. Would this step improve the 260/280 ratio or simply the yield?
Hi Rami,
From memory (I haven't tested this for a long time) you do get a different 260/280 ratio for partially solubilised RNA compared to completely solubilised. I believe that partially solubilised has a much lower 260/280 (
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