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#57851 qRT-PCR standard curve data analysis

Posted by UC Mol on 05 February 2010 - 01:28 PM in PCR, RT-PCR and Real-Time PCR

Hey,

I'm in a dilemma with my data analysis. My work is on transgenic mice that have isoforms of creatine kinase knocked out. I'm trying to quantify a few genes of interest (RYR complex, SERCA2) in the sarcoplasmic reticulum of murine cardiomyocytes. I have too many samples to do them all on one plate (96 well). So, obviously I will have to run a separate plate and thus a second standard curve. Here lies the problem: when comparing the same gene's data values between two plates, I have often run into different PCR efficiencies and correlation coefficients. For example, one plate may have a PCR efficiency of 97% while another may have 85%. This difference in standard curves efficiencies changes the values of my samples. I allow the MyIQ system to automatically calculated threshold to produce the best correlation coefficient (instead of doing this manually). So all in all, the values for samples in a group (experimental or control) are largely different from each other depending on the plate even though they have similar Ct's. Is it possible to apply one standard curve to all samples and plug in the Ct's into the linear equation to determine the SQ (starting quantities)??

FYI - I'm using MyIQ with SYBR green Rx mix.



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