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what Ct values are considered ideal? too high?


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#1 medstudent1

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Posted 26 March 2009 - 12:24 PM

hey all,
i am doing some rt-pcr experiments using taqman probes. i am differentiating 3t3-l1 pre-adipocytes to generate gene expression profiles for ppary, cebpa, and pref-1 (all well known in the literature). my ct values range from 20-35 depending on the gene and the time point. i also have a few unknown genes that i am examining to see if they play a role in adipogenesis and my ct values for these genes range from 30-35 depending on the time point. i am wondering if these ct values are too high? ideally i would like to shift my curves to the left so my ct values are lower but i am already using a lot of cdna in each reaction and it would be difficult to use more than i am at present. (by the way, when i select my threshold for the ct values, they all cross the amplification curve in the linear range)...

any advice would be great....

#2 pcrman

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Posted 26 March 2009 - 09:08 PM

What is the Ct value for your housekeeping gene? If the expression of GOI is very low you have to use more template and make more cDNA by RT reaction.




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