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Data analysis question: issues with gene expression's Ct difference


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#1 Osbourne

Osbourne

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Posted 29 April 2009 - 08:17 PM

Hi.
First of all, I'm a newbie in Real-time PCR, so forgive me for my prosaic questions.
I would like to do relative gene quantification based on GED formula (http://www.springerl...22/fulltext.pdf). Do you know this method?
My goal is to verify the expression pattern of a gene X in a starvation condition. So I have prepared three independent bacterial cultures (biological triplicate) in contrasting conditions : starvation (S) and non-starvation (NS). After, I have done the Real-time PCR, setting up reaction triplicates (technical triplicates) for the reference gene and gene X in each condition of every culture. It is not clear to me how to proceed from this stage: do I calculate the mean Ct and mean Efficiency of the technical triplicates, then I apply this values to rER (relative expression reation) formula? Or, do I calculate the mean rRER of all nine rER (three PCR of every culture)?

I don't know... Any suggestions?

Thanks.




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