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Efficiency correct fold change -- use all or none?

RT-PCR qPCR efficiency

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#1 jwag

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Posted 01 May 2014 - 10:36 AM

Hi all,

 

I'm currently analyzing RT-qPCR data and trying to use efficiency-corrected ddCt method (Fold change=E^ddCt) between 10 very different samples.  My problem is that the starting template numbers are very small, resulting in Cq values often >30.  In order to figure out efficiency slope, you need at least 3 data points of 10-fold dilutions.  Since I ran my samples for 40 cycles, this makes calculating efficiency slopes impossible for some of the samples with the lowest starting quantities.

 

So my question is, if I have some samples with acceptable efficiency slopes but others without, is it still appropriate to use this method?  I would have to assume that the ones without a calculated slope to have efficiencies of 2 or 100%, which is not likely true, and I'm afraid of biasing my data.

 

Has anyone else had this problem or had any thoughts? Thanks!

 

Josiah



#2 Trof

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Posted 02 May 2014 - 07:58 AM

I don't thing you need three 10-fold dilutions. That only makes a wider reference range which is beneficial.

 

You need at least a 3 data points for calculating efficiency, but doesn't need to be 10-fold if you can't get a good data.

In such cases we make 5-fold dilutions.

Anyway samples would probably fit in the dynamic range which is more narrow using 5-fold dilutions, samples also can't have valid data after some >35 cycles.

 

Running more than 40 cycles in case of low abundancy genes is also better, not because you would get any usable Ct after that, but for the late samples you will be able to see a curve reaching a full plateau.


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