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gDNA pcr product as standard for absolute quantification?


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#1 starvingstudent

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Posted 27 October 2009 - 06:42 PM

Hello all,
Can a pcr product of my gene of interest be used as qPCR standard instead of a plasmid for absolute transcript quantification?
Thanks!

#2 tea-test

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Posted 27 October 2009 - 11:14 PM

you mean for absolute quantification of mRNA copy numbers? No, you can't use it and to be accurate you also cannot use plasmid DNA because you don't have an adjustment for the reverse transcription efficiency. It is possible to use in vitro transcribed RNA of your GOI and reverse transcribe this RNA under the same conditions as your sample RNAs.

I think there have been discussions on the bioforum about that.
tea-test: The artist formerly known as Ned Land




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