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Pfaffl Method for relative - (Aug/28/2013 )

Dear all,

 

I want to ask about Pfaffl model for relative quantification. I use the formula:

ratio = E target^(dCt target)/ E reference ^(dCt reference).

Lets say I use normal cells for the calibrator, so the dCt is Ct differences between the normal cells and the samples, right? So what is the result? Is it n-fold relative to reference gene or is it n-fold relative to normal cells?

 

And I got question about the calibrator. If I run an experiment to comparing pre- and post-treatment expression for 10 patients (each patient has both pre- and post-treatment tissue, so in total 20 samples), is it better if I use the pre-treatment tissue (tumor tissue) or normal tissue as the calibrator? 

 

Really need your help through this. Thank you :)

 

Cheers,

 

dee

-detriar-

Lets say I use normal cells for the calibrator, so the dCt is Ct differences between the normal cells and the samples, right? So what is the result? Is it n-fold relative to reference gene or is it n-fold relative to normal cells?

What is the difference between the reference gene and the normal cells? For this sort of experiment the levels will be assessed relative to a gene from the cells, this could be any one of a number of reference genes that you have assessed and found to be consistently expressed in your tissues of interest.

And I got question about the calibrator. If I run an experiment to comparing pre- and post-treatment expression for 10 patients (each patient has both pre- and post-treatment tissue, so in total 20 samples), is it better if I use the pre-treatment tissue (tumor tissue) or normal tissue as the calibrator?

It depends on what you are looking at - if you are looking at gene expression affect in the tumor cells then you need to compare with the tumor tissue, but if you are looking for a change in the cells that is indicative of the tumor itself, then you compare to normal tissue.

-bob1-

Thank you  for the reply, it's very helpful smile.png. I got confused about that, but from your explanation it makes sense now.

 

Btw can you suggest me if it is okay to run the GOI gene and Ref gene not in the same experiment? What if the annealing temp between those two are different? 

 

Cheers,

 

dee

-detriar-

You could so long as you have some method of inter-plate comparison, this could be a DNA that you know the CT value for both GOI and reference.

-bob1-

I see, so it's required more tasks, that's why it's very important to set the same annealing temperature between the GOI and Ref gene from the first, eh? I'll keep that in mind if I'm about to set a new primer pair.

 

Thank you very much, 

-detriar-