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

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Postby pcruser » Thu Dec 10, 2009 3:39 pm

Hi Mikael
Just a quick question, I assume in the nested design demo that the IPC calibrator used was a single sample in triplicate ( per probe ieFAM is only FAM used)that was averaged...the same used on all plates (ie 1-36,37-72 in your expt--- same as for example avg triplicate sample #1 GeneA plate1,avg triplicate sample#1GeneA plate #2) and that the difference you saw in the IPC of plate #1 and plate#2 when run against its own plate is enough to calibrate each indiv plate to be compared to each other? Or do i need to take an avg of IPC on all plates or compare the avg IPC and compare between plates to be used ??
Also when using the TAQman primers designed to be pcr efficient, on MG and RG throughout the expt,do we need to pre-process the PCR effeciency?
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Joined: Wed Dec 09, 2009 4:07 pm

Re: IPCs

Postby Mikael Kubista » Sat Dec 12, 2009 10:26 pm

Yes, you can use a single IPC (preferably measured in triplicates minimum) for each instrument channel used.

Normalization can be performed by subtracting the Cq of the IPC from the measured Cq’s in the same plate. This, however, typically gives rather low corrected Cq values. GenEx therefore adds the average Cq of all IPC’s to the corrected Cq’s, which offsets the corrected Cq’s to the original magnitudes. This is for convenience mainly. Mathematically, the two approaches are equivalent.
The two main factors that affect PCR efficiency are:

1) The assay performance
2) Interference from components in sample matrix

You are referring to the first effect only. Whether correction is important or not depends on the analysis you are doing and what results you are calculating. Easiest is to to test the effect of PCR efficiency for your particular experiment. Analyze data once without correcting for PCR efficiency and then repeat the analysis assuming certain PCR efficiencies. Its good strategy to test the effect of the GOI and RG PCR’s being inhibited to the same degree (i.e., both being 85 %), which often is the effect of matrix interference, and also having different efficiencies (85% and 90 % respectively). This exercise answers the question for your particular case.

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

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