qPCR fold change

[Mag]

Hi,

Could anybody help with the following problem..?

I`m trying to validate my microarray results and qPCR data shows much higher fold change to what was calculated from microarrays....(i.e microarray fold change between two samples was 1.9, from qPCR 700). The tendency (up - down regulation) is maintained, it`s just this fold change that is so much different.

I use 7 samples (its time course experiment) and firstly Ct values were transformed to quantities according to geNorm relative quantification protocol. Then, samples were normalised according to normalisation factor calculated as a geometric mean of two houskeeping genes. And the experiments were run in triplicates (3 independednt pools) of course each sample was run in triplicates as well.

If anybody ever had similar problem and could give me any hints would be great. Is this kind of result normal?

Thank you for any help....

[amicia]

Hi,
I think it's normal! That's cause qPCr is more sensitive than microarrays. Four years ago during a congress there was a relator reporting more or less the same problem than you. He concluded it was a problem of sensitivity.

[Mag]

Hi,

Thank you very much for your reply.
That gave me a hope, that data is OK

I thought so too, but what concerned me was that bascically in all of the published papers I saw, the fold change of microarrays and qPCRs was approximately similar. There were one or two only where the actual fold change detected with qPCR was over 40. Maybe I`m missing somthing...?

[amicia]

Hi,
I'm not an expert in the field of microarrays and I don't know the procedure and the functions You used to detect Yours microarray's results. Anyway, for the qPCR I think You had used the correct approach (basically the best choice is to use three or more housekeeping genes, but I don't think this is the problem in Your case).