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question about pcr amplification efficiency?


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#1 kedar

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Posted 11 August 2011 - 07:32 AM

Dear all,

I am trying to see the pcr efficiency between target gene and control house keeping gene. because if i am right, they must have same efficiency for delta-delta ct to be used.
Here are my Ct for gene of interest- 31, 33, 35 (for 1:2, 1:4, 1:8 serial dilution of cDNA)
Ct for Gapdh- 18, 19, 20 (for 1:2, 1:4, 1:8 serial dilution of cDNA)

You can see that it takes 2 cycle each for my gene of interest to double(31-33, 33-35). While it just takes 1 cycle more (18-19-20) for GapDh.

Does this mean the pcr efficiency is not same between target gene and house keeping gene? so delta-delta Ct method cannot be applied?!

In this scenario, what can i do to get the same 1 cycle each for my gene of interest?

thanks a lot.

#2 Archie

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Posted 02 November 2011 - 04:39 AM

Hi,
I think it is worth mentioning that PCR efficiency is not the same between different amount of templates.
E.g. you may get doubled amplification within 1 cycle for higly abundant target DNA at Cq of 20 but for less abundant DNA (Cq of 30) it may take more than one cycle to double your target. This can have several reasons: primer dimers, less polymerase available at a later cycle, design of primers etc.

I would recommend you to do several ten-fold for your Gapdh:
start 1:2, then dilute 10x with 5 µL cDNA and 45 µL water (1:20), dilute this 1:20 10x again (5 µL cDNA + 45 µL water = 1:200) and so on until you have at least 5 different dilutions. If the slope between each of these dilutions is about -3.32 cycles then your efficiency for the reference gene is 100%. Slopes of -3.1 to -3.6 are generally considered acceptable.

Try to do the same for your sample with higher amount of cDNA maybe (start with undiluted sample, increase the volume of sample in the reaction).
Would be good if your sample with the gene of interest would at least have a Cq in the 20s.

I'm not doing much relative quantification but what I've read so far is that efficiency of reference gene and your target can differ quite a lot due to different primer design, abundance of target etc.




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