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Real time ddCt experiment reference setup for plates

ddCt reference control plate

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#1 assembler01



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Posted 29 May 2014 - 08:08 AM

In my comparative Ct study on an applied biosystems 7500fast real time machine, I ran a plate first with all of my RT-qPCR samples for beta-actin (reference gene). I do each sample in triplicate. I have 6 groups of 5 animals each. They will all be compared the the first group, C57Bl/6 no treatment. Then I ran the second plate with my target gene (gene1). I included 2 of the 30 samples with beta-actin on the second plate (one is my calibration sample [a B6 CD] to run on all plates and one was a failed beta-actin reaction I had to redo). The software will not calculate the gene expression (2^ddCt or other method) for gene1 because it can't seem to figure out I want it to use the first plate as the reference/housekeeping gene. I had to go back and do the calculations manually to try it this first time, but is there any way to get the program to calculate the gene expression of all my plates for me? I will also be running several more genes as well as one or two more reference genes if I still have enough sample in the end. If I have to run the reference genes and target gene all on the same plate, I will need an infinitely large plate and a robot to load them for me, as well as a ton of sample.

Thank you very much.

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