IPC calculation and normalization to PBMC pool

Here we discuss the use and processing of inter-plate calibrators

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IPC calculation and normalization to PBMC pool

Postby jchrist » Mon Jan 23, 2012 11:04 am

Hello,
I have a question regarding the calculation of IPC. We have a experimental design where we want to analyze gene expression in lymphocyte subset isolated from human PBMCs. Because of a high sample number, we run each target on two different 96-well plates. In order to normalize run-to-run variation we have used a cDNA pool from human PBMCs as standard samples on each plate.

Now, we would like to have the expression value to reflect the expression of a target in relation to the PBMC pool, instead of arbitrary units. We thought we could modify the formula for calculating IPC-normalization of the Ct-values of the targets just by subtracting the Ct value of IPC-standards from the same plate from each target Ct value run on the same plate, and then leave out the addition of the mean form all the IPC-standards.

- So my question is, if this way of calculating IPC-normalization is safe, or am I missing any important details which could result in a bias?

Regards Jeppe
jchrist
 
Posts: 1
Joined: Mon Jan 23, 2012 10:44 am

Re: IPC calculation and normalization to PBMC pool

Postby Mikael Kubista » Tue Jan 24, 2012 3:50 pm

Jeppe,
If I read you correctly, this is in fact the way leading qPCR software such as GenEx does. I suggest you download the free (trial) version from www.multid.se and compare!

Good luck
Mikael Kubista
 
Posts: 455
Joined: Tue Jul 01, 2008 12:28 pm


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