RNA concentraion - (Nov/09/2006 )

During the RT-step (thermoscript), I used 10ug of RNA, eventhough protocol says 10ng-5ug.

If I use more RNA (g), what kind of disadvantage I will get???


-jae

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Dear jae,

A too high amount of template RNA may reduce (to inhibition, even) performance of RT-PCR. I think 10 ug would be not such a big probleam, but grams??? I can see you get lots of RNA (or you've mistaken the units?)
RT is usually a low-efficiency reaction due to several reasons, including the reverstranscriptases behaviour. An optimal template/primers ratio, at least, is desirable.
If you are not convinced, try to do the PCR with your cDNA and see the result... smearing will occur, at least, not to mention you may have the PCR less efficient, inhibited even

You're wellcome

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If you want to use more RNA just do more reactions and pool them afterwards.

I can't think of any RT-PCR application that requires g of RNA - after all the whole point of RT-PCR is so you don't need to isolate huge amounts of RNA.

Daniel

Longer ABI sequencing traces

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