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Ct values - (Nov/19/2012 )

I am using mice GAPDH as reference and IL2 gene as my target(Taqman gene assay). il2 has a very low copy number and showed CT values ranging from 35-38. and GAPDH CT values ranges from 26-28. I used 20ng of cDNA. So I increased the cDNA concentration to 200-300ng and observe that Ct value for il2 comes down to 30-32 and GAPDH Ct value to 18-22. So can I use 200-300ng of cDNA for IL2 and 20ng of cDNA for GAPDH? Any suggestions or recommendations are highly appreciated!

-cell_bee-

I think it should be fine as long as you use this same amount of template input for every sample that you are making a comparison with. The expression is all relative anyways.

-CJB-

Housekeeping gene should be idealy from the same cDNA as the target gene, otherwise you are actually measuring different samples. The same is true for the dilutions of the same cDNA. In case there is different concentraton of inhibitors, you could get uneven ratios for undiluted and diluted cDNA.
I would try to limit GAPDH primer concentration to get higher Ct or use a different houseekeping gene that has a lower abundance.

-Trof-

Thank you! I shall try using different endogenous control.

-cell_bee-