This topic could probably have it's own StackExchange, there is so much info and trial and error done with rtPCR. Here's my 2 cents:

Your adjusted calculation does not normalize your data, it just subtracts the 'background' or control. For expression studies this is not good enough. Normalizing data for gene expression must involve setting it to a scale of another gene whose expression should be not only present but consistent between your treated and untreated samples. House keeping genes (you are probably using GAPDH) make good candidates for this because their expression rarely changes. If you don't normalize than you don't know if your your expression is actually changing, or if it's simply due to a larger input of mRNA. However it must be shown that the housekeeping gene's expression is also not changing, this is usually accomplished using a different sample set. Take if from me, I've done a lot of this and this step is one of the most important in your experiment. Any reviewer who knows what he's doing will reject a paper using rtPCR without the proper statistics.

I have had a lot of success using Applied Biosystems StepOne software to provide you with accurate Ct values. Generating the variable is complication, use software to do it right every time:

http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf

What method/system are you using?

Real time PCR normalization algorithm
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