Usually, these assumptions do not hold in practice but, strikingly, in most, studies this fact is entirely ignored. In our studies we rely therefore on nonparametric alternatives17,35 (Figure 2d): Wilcoxon’s rank sum test is based on the ranks of the replicates rather than on the actual signal values. This test (and other tests based on linear rank statistics such as the van der Waerden test) is preferable to the parametric

t- tests if the distributional assumptions cannot be proven to be Gaussian. PF-562271 clinical trial Furthermore, for “noisy” data this test, yields more robust results since it. is less sensitive against outlier Inhibitors,research,lifescience,medical values. For larger sample sizes, ie, >25 replicates, we can Inhibitors,research,lifescience,medical approximate the P value of the Wilcoxon rank test by the standard normal distribution. However, most practical applications will be based on a rather smaller number of observations (sample sizes in the order of 4 to 12). Therefore, those P values must, be calculated exactly This can be done using a recursive method.36

If several different experimental Inhibitors,research,lifescience,medical conditions are screened (for example different, time points after medical treatment), then each gene expresses a certain numerical profile across these conditions. Clustering algorithms are explorative statistical methods that group together genes with similar profiles and separate genes with dissimilar profiles, whereby similarity (or dissimilarity) is defined numerically by a pairwise (dis)similarity function such as Euclidean distance or Pearson correlation.37-40 Inhibitors,research,lifescience,medical Hierarchical clustering can be combined with a colorcoded representation of the signal values (the expression patterns) and visualized in the form of a dendrogram. Clustering is a very intuitive way of visualizing data, but it. should be pointed out that, the dendrogram is strongly dependent, on the parameters chosen for cluster analysis. Inhibitors,research,lifescience,medical Thus, each clustering process should undergo decent validation.41 Associated groups of genes42 are usually further investigated, for example for common binding sites in the promoter

sequences of the genes or for common functional content.43 The major result, of the explorative analysis is essentially a. list of potential marker genes relevant, for the disease or treatment under analysis. Since microarray data is errorprone, this list contains a lot, of false positives. Thus, further filtering to steps are commonly included in the analysis. Recent, methods therefore aim at, the correlation of the gene expression profiles with complementing sources of data such as pathway annotation, gene ontology (GO) categories, sequence analysis, clinical data, etc.44-46 Genes do not. act as individual units; they collaborate in overlapping pathways, the deregulation of which is a hallmark for the disease under study New bioinformatics tools have been developed that judge gene expression changes in the context of such pathway analysis.