Inter-run calibration plus control sample

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

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Inter-run calibration plus control sample

Postby oyster_lab » Mon Jul 28, 2014 11:42 am

Hey everybody,

I'm having some issues with my calculations for relative expression. As I have several samples and I need to run them in multiple plates. I have then included a inter-run calibrator (IRC) in all my plates - Single cDNA sample present in all my plates (in triplicate) and that has been tested for all my target and reference genes. In my case I want to normalize my data to the IRC and then normalize them again to the control sample of my experiment. Could you please have a look at my workflow to see whether it makes sense:

Step 1: Normalization of the Cq values for each target gene in relation to a reference gene (or a combination of reference genes). In this case I would calculate the DeltaCq, which would be (Cq of target gene - Cq reference gene) for each of my samples, including my IRC.

Step 2: Inter-run calibration: Normalization of the DeltaCq of my samples in relation to the DeltaCq of my IRC. This would be (DeltaCq sample - Delta Cq of IRC), which would result in a DeltaDeltaCq.

Does my calculation make sense until here?

Potential set 3: From here, I still would like to use an extra calibrator, which would be the control sample of my experiment. For example, I'm testing animals infected by a pathogen at different time points, and I want to compare their gene expression levels to non-infected animals. Would that be possible if I need to do an IRC as well?

Thank you very much for your help!
oyster_lab
 
Posts: 1
Joined: Mon Jul 28, 2014 8:53 am

Re: Inter-run calibration plus control sample

Postby Mikael Kubista » Mon Jul 28, 2014 5:06 pm

You can, although its more common to remove the variations between the plates in a separate first step by simply subtracting the mean of the IPC's (or IRC's as you call them). Check the GenEx manual for details; its available here: http://www.tataa.com/products/product-downloads/.

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


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