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Real time PCR, issue normalization

Real time PCR, issue normalization - PCR - Polymerase Chain Reaction Forum

Real time PCR, issue normalization - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 04-19-2013, 11:29 AM
Pipette Filler
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Default Real time PCR, issue normalization



Hello all,

I am new to the forum and posting because I can't seem to find anyone in my lab which does work on qPCR.

I am interested in getting some values of expression level for one target gene. All I want to do is to get a d(ct) value which will represent the level of expression for all my 400 samples so I can see how the expression differs over time and space bz doing some mixed effect model.

I have one target gene an three reference genes, the efficiency of the genes vary by more than 10% so I thought about using the pfaffl method to calculate the d(ct) value. I used the following equation : dct= efficiency target ^ct taget / eff ref ^ dct ref.
However, I realized quickly that there is an issue with my results.
Samples which have the highest expression value (lowest ct value before normalization) ends up with the highest d(ct) values and therefore when I try to plot the values for the manuscript, the lowest expressed samples seem to be the highest.

I hope you follow me, it is a bit hard to explain, all I want to say is that my dct values are kinda reversed and the plot does not make sense and is much harder to explain than it should.

Do you know if I made a mistake or if there is smtg I can do to make my results and graph better?

Thanks a lot everybody

CLaire
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