Hi friends,
I am using the SuperScriptFirst-Strand Synthesis System for RT-PCR, from Invitrogen (#11904-018) and, unfortunately in our kit the RNAse H vial is lost. Of course I can just buy a new kit however I was wondering if I can use RNAse A instead H, because we have it in our lab?
I tried to dig into this issue, and understood that I can NOT use the A (so I just frozen my product without that 20' RNAseH treatment... until better times), however might you have some alternative opinion?
Thank you
RNase H should digest RNA selectively from DNA:RNA hybrids, but generally this step is unnecessary for RT-PCR since Taq DNA pol is a DNA (not RNA) dependent polymerase. Thus, during the initial denaturation step of PCR (typically @ 94 °C) the hybrids should split up and the annealing of primers to this neglectable amount of RNA would be irrelevant, since the enzyme will not extend a DNA strand on it.
What Dmitry says is true, there is the old acid hydrolysis method used for Feulgen's reaction for instance, which takes advantage on the higher lability of RNA due to its extra OH under acidic conditions at 65 °C (typically 10 min).
IMHO, I would not bother doing any sort of trick and would proceed straight to PCR unless you suspect that the RNA is for somewhat reason a real problem.
Best of luck
Hi, Mark!
There is an RNAse-free method to degrade RNA. You need to heat RNA sample to at least 65C in the presence of Mg2+ ions. Since Mg ions are presented in the kit just heat your mixture. RNA degrades because it has additional -OH group on the sugar, DNA should be stable. I think single stranded RNA should be degraded in RNA-DNA hybrids too. You may found details of the mechanism and effect of other ions on the RNA in the paper.
Good luck!
RNase H should digest RNA selectively from DNA:RNA hybrids, but generally this step is unnecessary for RT-PCR since Taq DNA pol is a DNA (not RNA) dependent polymerase. Thus, during the initial denaturation step of PCR (typically @ 94 °C) the hybrids should split up and the annealing of primers to this neglectable amount of RNA would be irrelevant, since the enzyme will not extend a DNA strand on it.
What Dmitry says is true, there is the old acid hydrolysis method used for Feulgen's reaction for instance, which takes advantage on the higher lability of RNA due to its extra OH under acidic conditions at 65 °C (typically 10 min).
IMHO, I would not bother doing any sort of trick and would proceed straight to PCR unless you suspect that the RNA is for somewhat reason a real problem.
Best of luck
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