A technical problem arises from the fact that a rigorous distinction between bands which are affected by the treatment and those which are not affected is only possible after the normalization, yet it is necessary to identify at least some unaffected bands for the calculation of the normalization factor.
For the calculation of experimental normalization approaches, the raw spectra were subjected to a Tophat baseline subtraction [ 14] and exported as xy-values to text files.
The median value of all intensities in the spectrum is used for the calculation of the normalization constant: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ff = median\left({{y_i}} \right).
The authors calculated their normalization factor from the intensities of bands found to be expressed constantly during a time-course experiment while we automatized this process by calculating intensity ratios for all bands, sorting the bands by ratios and using bands from the 25%75%75% quartile for the calculation of a normalization factor.
Six scaling constants, one for each of the six normalizing proteins, were used in the calculation of the normalization factor.
In each case, the two most stable reference genes were sufficient for accurate normalization, since inclusion of a third gene had little impact on the calculation of the normalization factor (V n/V n+1 below 0.15).
geNorm software was used to establish the two most stable internal control genes (MRPL19 and PUM1) from a group of four endogenous controls (ACTIN, GAPDH, PUM1 and MRPL19), followed by the calculation of the normalization factor for each tissue sample (Table S2 in Additional file 1).
All the reactions were carried out in duplicate, and fold changes in gene expression were determined by using the formula 2-ΔΔ C t. geNorm software [ 32] was used to establish the two most stable internal control genes (MRPL19 and PUM1) from a group of four endogenous controls (ACTIN, GAPDH, PUM1 and MRPL19), followed by the calculation of the normalization factor for each tissue sample.
As with EPN, six scaling constants were used in the calculation of the normalization factor.
The gene rpoB was used as an internal standard for the calculation of expression levels and normalization.
The qbasePLUS software (Biogazelle, Ghent, Belgium) was used for the calculation of the relative quantities using RPP30 for normalization (D'haene et al. 2010).
