Assessing Signal-to-Noise in Quantitative Proteomics: Multivariate Statistical Analysis in DIGE Experiments - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 45

9. By performing the prealignment, the SameSpots approach

enforces no missing values by having all features analyzed with
the same spot boundaries. But this is at the expense of loosing
information from features that are not well resolved on all gels
(feature crowding).

10. SameSpots provides for the visualization of as many principle

components as can be detected as contributing to the variance.
Any of these principal components can be visualized in pairwise
2-dimensional displays.
11. When only two conditions are present (e.g., mutant vs. wild
type), applying an ANOVA or Student's t -test to fi lter the data
should necessarily designate PC1 as organizing the samples by
condition, although in these cases this test may still be useful
to assess the clustering within each classifi cation to determine
if the biological signals are uniform or infl uenced by other
dimensions of principal components.
12. Dye bias is controlled for by labeling half of the samples with
Cy3 and the other half with Cy5 from a given experimental
condition. The groups separated by PC1 in Fig. 3b each have
two members from each experimental condition, thereby
negating any changes due to dye bias coming through a statis-
tical test between biological groups.
13. Using the two-dye system, dye bias does not contribute to the
experimental variation if the internal standard is always labeled
with one dye (Cy3) and the experimental samples are always
labeled with the other (Cy5).

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