Not enough cDNA for qRT-PCR - how to increase cDNA yield or save cDNA? - (Feb/20/2006 )
hi,
This means that RNA can be degraded very easy? so, is it possible that during DNase treatment that involve incubaqtion during 30 min at 37C, and then inactivation at 65C RNA is degraded? also is it posible that RNA can be degraded during RT reaction? How can be store the RNA to preserve it? how much RNA can be degraded at -80?
thanks
-operon-
hi,
yes, RNA degrades very easily, which is why its always suggested to store it at -80 and to RT straight away if possible.
I am not sure about whether it can degrade during Dnase?
-smurray-
QUOTE (operon @ Mar 7 2006, 10:09 PM)
hi,
This means that RNA can be degraded very easy? so, is it possible that during DNase treatment that involve incubaqtion during 30 min at 37C, and then inactivation at 65C RNA is degraded? also is it posible that RNA can be degraded during RT reaction? How can be store the RNA to preserve it? how much RNA can be degraded at -80?
thanks
This means that RNA can be degraded very easy? so, is it possible that during DNase treatment that involve incubaqtion during 30 min at 37C, and then inactivation at 65C RNA is degraded? also is it posible that RNA can be degraded during RT reaction? How can be store the RNA to preserve it? how much RNA can be degraded at -80?
thanks
Hi Operon-
You might find this technote by Ambion helpful. It discusses how most DNase is contaminated with RNase. Recently I did an experiment where I bioanalyzed my RNA before and after DNase treatment along side a control that went through all the same conditions except for the DNase I. My RNA was noticeably degraded by the DNase treatment, but the controls were not. I informed the supplier and received a new shipment of DNase that is actually OK and does not affect my RNA quality.
Sorry, I can't really help with the rest of your questions, except to say that RNA is always susceptible to degradation and that keeping it cold (store at -80, thaw on ice, etc) just helps slow the degradation down. I can't imagine that the RT reaction is that bad, since it is widely used and it works. But these thoughts are just based on my limited experience.
-soluene-