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Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A

;ΔCT value was then calculated by subtracting the ΔCT value for the sample immunoprecipitated with Pdx1 antiserum from the ΔCT value for the corresponding control sample immunoprecipitated with normal rabbit serum. Fold differences (Pdx1 ChIP relative to control ChIP) were then determined by raising 2 to the ΔΔCT power. The equation used in these calculations is:

Fold-difference
(Pdx1 ChIP relative to control ChIP) = 2[ΔCT(control) Δ CT(Pdx1)]

where ΔCT = CT(immunoprecipitated sample) CT(input)

For every promoter fragment analyzed, each sample was quantitated in duplicate on at least two separate occasions, and from at least two independent immunoprecipitations, to give a minimum of four independent quantitations of each sample for each promoter. Quantitations were corrected to account for 10% inputs. Mean SD values were determined for each fold difference, and these values were subsequently used in two-tailed paired Students t tests to determine statistical significance. A melt-curve analysis was performed for each sample after PCR amplification, to ensure that a single product of expected melt-curve characteristics was obtained.


Results and Discussion
Previous studies have documented that Pdx1 binds in vitro specifically to promoter elements containing the TAAT motif (Watada et al. 1996, Ohneda et al. 2000). However, none of these studies have examined directly the specificity of promoter binding in vivo, where the complexity of chromatin structure might physically hinder accessibilit
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