ct Validation test - using GAPDH endogenous (Nov/01/2006 )

hi all.

need expert advises.

I am doing the validation test to see if I can use relative Ct study to study the gene expression level.

I used GAPDH as endogeneous control, ran a series of concentration of DNA template vs my target gene. I ran non template control (NTC) too.

Firstly, I got values for all my GAPDH samples including the NTC.
Secondly, the values for my GAPDH were all quite the same although i used diferent conc. of DNA.

Please help...

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wow, that's a mess

did you try a gel or a dissociation curve? are you sure gapdh is what you're measuring? what do you see with the NTC? could you please provide more detail?

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ya, a big headache indeed!
I ran the dissociation curve. NTC samples shown a peak at temp similar to other samples. It's gapdh, no mistake that I am measuring cos the primer set is shared by lab mates. They got no problem with it.

that's y i guess might be my cell line- PC12

what other control can i use then?

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I think you may need to fix the current problem..you may see the same thing if you switch to another primer set

I think it's a problem that you are getting a peak in your NTC. what concentrations of primer have you tried? it may be dimers...how does this peak compare to what your labmates have seen, on the dissociation curve?

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