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Will I need interplate calibration for my RT-PCR experiment?


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#1 cells

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Posted 02 April 2011 - 07:57 PM

Hi, I have a quick question regarding interplate calibration. I have never used interplate calibration before so I'm a bit confused if I do need to use it for the following setup or not. Basically, my experiment is: I have two genes that I am interested in at four different time points. I have two reference genes Actin and GAPDH.

So I'm wondering if I run individual timepoints with my housekeeping genes on the same plate, will I be able to compare between different time points run on different plates without worrying about interplate calibration?

So basically, I will run timepoint 1 on one plate with my two genes of interest along with the housekeeping genes; timepoint 2 will be run the same way on a different plate and so on. If I normalize my genes of interest on each plate with the housekeeping genes, I will get the normalized relative fold change - can I compare these fold change with the fold changes I get from the other plates. I would think so since I'm running the housekeeping genes on the same plate and therefore in the end, I will just be comparing the normalized changes within each plate so I don't think I have to worry about any other conditions that may affect results between different plate....but I'm a bit unsure so if anyone has any suggestions it would be great.

Thanks.

#2 tea-test

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Posted 04 April 2011 - 12:49 AM

Yes, you will need interplate calibration. doesn't matter if you also have the HKGs on the same plate.

A possibility to avoid this:

Put all samples that you want to compare on one plate, that means put all timepoints from gene A on the first plate, gene B on the second and so on.... (also for the HKGs). But this only works if you don't have so many samples that they don't fit on one plate.
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#3 PostDocTrauma

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Posted 05 April 2011 - 12:43 AM

I would have thought that if you have converted the same amount of RNA to cDNA for all the timepoints then you wouldn't need to run an interplate calibration.

We normally convert 100ng/100ul and add 1ul for each reaction.

#4 Trof

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Posted 05 April 2011 - 01:24 AM

I would have thought that if you have converted the same amount of RNA to cDNA for all the timepoints then you wouldn't need to run an interplate calibration.

We normally convert 100ng/100ul and add 1ul for each reaction.

No, you do need it. The cycler isn't THAT reproducible, every run can have a little difference, in temperature cycling, in fluorescence, in everything. You don't need it only when you run all the samples you want to compare together on one plate/run, as tea-test said. Other genes can be on different runs, because you don't compare genes but samples.

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