2D DIGE Analysis of Protein Extracts from Muscle Tissue - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 164

one-way ANOVA, and it is used to test for differences in
standardized abundance among experimental groups; the test
is accompanied by Tukey ' s test (post hoc test) to get an indica-
tion of which group is different. The two-way ANOVA is used
to analyze two conditions in an experimental design in which
two independent factors are taken into consideration. It calcu-
lates the signifi cance of the difference between groups with the
same condition 2 and different condition 1 (indicated as two-
way ANOVA condition 1) and the other way around (two-way
ANOVA condition 2). The two-way ANOVA analysis also
calculates a signifi cance value of the mutual effect of the two
factors (two-way ANOVA interaction). Signifi cantly changed
proteins having a p value <0.01 are typically considered.

- Principal Component Analysis ( PCA ). This analysis is essen-

tially a method for reducing the dimension of the variables in a
multidimensional space getting a simpler view of the proteins
and the spot maps in the data set. It is thus possible to detect
outliers in the data set and to identify spot maps that have simi-
lar expression profi les.

- Pattern Analysis . This process consists of algorithms that can

help to fi nd the subsets of the data that show similar expression
patterns. There are different types of pattern algorithms: one
of the most important is Hierarchical Clustering , a method
that combines or splits data pairwise and generates a tree-like
structure called dendrogram. Protein and spot maps with simi-
lar expression profi les are grouped together.

4. Notes

1. The cells or tissue should be disrupted in such a way as to mini-

mize proteolysis and at low temperature with a minimum of
heat generation. There are different disruption methods, both
mechanical and chemical ( 14, 15 ). Gentle lysis methods
(osmotic lysis ( 16 ), freeze-thaw lysis ( 14, 17, 18 ), enzymatic
lysis ( 19, 20 )) are generally employed when the sample of
interest consists of easily lysed cells. More vigorous lysis meth-
ods (sonication ( 21, 22 ), grinding ( 23-25 ), mechanical
homogenization ( 17, 26 ), glass bead homogenization ( 27 ))
are employed when cells are less easily disrupted (cells in solid
tissue or cells with tough cell walls).

2. Appropriate sample preparation is absolutely essential for good

2D DIGE results. The optimal procedure must be determined
empirically for each sample type. Ideally, the process will result
in the complete solubilization, disaggregation, denaturation,
and reduction of the proteins. It is important to remember