The ECL signal was quantitated using a scanner and a densitometry program (Scion Image; Scion, Frederick, MD). Background pixel values were subtracted, the signal was normalized to the amount of actin and the normalized band intensity values were plotted.
Finally, an approach based on tolerance intervals and accuracy profiles was applied in order to thoroughly validate the method in a range of concentrations comprised from 3 to 15 µg mL−1 using normalized band intensities.
To distinguish SMN2 (line 89) from SMN2 (line 566), we performed the above PCR using a limited number of 20 cycles and normalized band intensity to that obtained from PCR-based detection of the endogenous, single-copy gene, IL-2 (IL2-F, 5'-ctaggccacagaattgaaagatct-3' and IL2-R, 5'-gtaggtggaaattctagcatcatcc-3') [64].
The normalized band intensities were plotted against GF concentrations.
Quantification of normalized band densities indicated that DMBA significantly decreased LPS-induced NF-κB-DNA binding by 33% (p < 0.05).
Fingerprinting patterns were analyzed in the Image 3.10b gel processing program [ 55] to generate normalized band position files.
Normalized band powers from 5 ROIs (AC1, AC2, OF, DPFC, and PA) within the left and right hemispheres were averaged over hemispheres for each ROI and every patient separately, resulting in seven ROIs for 28 patients [Kahlbrock and Weisz, 2008].
a From the Ribonuclease Protection Assays (RPAs), mean fold-change was determined by normalizing the optical density band of the LPS-treated sample to the housekeeping gene GAPDH or L-32, then expressing it relative to the optical density of the normalized band from the vehicle-treated sample.
The quantification of the normalized band intensities revealed no significant increase of harmonin expression following co-administration of NB54 or PTC124 compared to single treatments (Fig S1B of Supporting Information) indicating no synergistic effect of the co-administration of both TRIDs.
The fact that induction factors for the vast majority of data points calculated from normalized band intensities were close to 1.0 (Fig. 5) indicates that the reproducibility of DDRT-PCR patterns was fairly good in this experiment, though we did not know which part of the differences is attributable to induction/suppression and which to random variance.
