3.5. Combined Methods
Another approach which combines quantification, loading control normalization and spatial normalization is the “SuperCurve”-based method NormaCurve, published in 2012 by Troncale et al. [22]. NormaCurve proposes basically an extended “SuperCurve” model composed of a non-parametric model to quantify relative protein expression from Hu et al. [16]. An additive extension takes into account sample effects and spatial effects. Compared to other “Super-Curve”-based models, this one allows a full and reproducible removal of spatial bias as it considers positive control and total protein stained arrays for normalization and spatial covariates for correction of the spatial bias, respectively. The resulting model was further explored to assess the reproducibility of control arrays between slides, the optimal number of replicate spots and the minimally required number of serial dilution steps, as addressed in the following section.