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Problem with the normalization of PCR Ct values

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Problem with the normalization of PCR Ct values

Postby Tzia » Tue Apr 30, 2013 12:41 pm

Hi!

I have some difficulties with the normalization of Ct values.. I do not have a calibrator, I just observe differences, thus, I just need a way to normalize my data and compare them with the endogenous control simultaneously.
I have read somewhere that one way for normalizing PCR data is to divide the Ct of the gene of interest by the Ct obtained from a housekeeping/reference gene (Ct inter. / Ct refer.). However, I am not sure how to interpret the resulting values. Is it true that the larger the ratio, the lower the expression of the gene of interest?
Additionaly, what about the 2^-ΔCt (ΔCt = Ct inter. - Ct refer.) ? What does this value mean? Provided that 2^-ΔCt = X, can I state something like: "The expression was observed to be X copies of the gene of interest mRNA / copy of reference gene mRNA" ? Finally, 2^-ΔCt values less than one means that the expession of the gene of interest is less than the expression of the reference one, whereas for 2^-ΔCt values more than one holds the opposite?

Any help is more than welcome!!!!
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Postby JackBean » Tue Apr 30, 2013 3:55 pm

You should not divide the CTs, because they are not linear. You have to subtract them and the difference will tell you how many times more copies there was.
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Postby bravebeaker » Wed May 01, 2013 1:12 am

in my case when I have a gene of interest and the standard gene (usually actin) I divide the calculated conc. in fmol of the gene of interest by the calculated conc. of actin.
about the ct values, the higher the value, less copies are there, if you make a standard curve you can see how the ct values get higher when you dilute your DNA more.
about the formulas of ct value differences, you should check the help section of the software you are using of the cycler's machine, it can explain many aspects for your interpretation.
hope that helps.
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Postby JackBean » Wed May 01, 2013 8:45 am

but without quantification he cannot calculate the amount of his GOI
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Postby Tzia » Wed May 01, 2013 8:48 pm

Hi!! Thanks a lot for your replies!

Probably I misunderstood the term "ratio". When dealing with exponents, division corresponds to subtraction...
So, I will subtract the Ct values to get the ΔCt. These values are inserted to the statistical package. After the statistical analysis is finished, I have to interpret them...
Provided that ΔCt=Ct reference - Ct target=X, what does this resulting value mean? The higher the value, the higher the expression ? In regard to formulating this, can I say something like "The expression was observed to be X copies of the gene of interest mRNA / copy of reference gene mRNA" ?
Finally if I want to present the data as 2^ΔCt, negative values mean down-regulation and positive values up-regulation?

If you could suggest any literature dealing with this issue, I would be very thankful....
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Postby bravebeaker » Thu May 02, 2013 5:36 am

Jackbean I agree with you, quantification of the pooled cDNA for the standard curve will result in knowing the conc. of the gene of interest.

Tzua, any paper dealing with the expression level of a certain gene using RT-qPCR would be suffice I guess.
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