Gene expression arrays in cancer research: methods and applications Ricardo R. Brentani, Dirce Maria Carraro, Sergio Verjovski-Almeida, Eduardo M. Reis, E. Jordão Neves, Sandro J. de Souza, Alex F. Carvalho, Helena Brentani, Luiz F.L. Reis  Critical Reviews in Oncology / Hematology  Volume 54, Issue 2, Pages 95-105 (May 2005) DOI: 10.1016/j.critrevonc.2004.12.006 Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 The influence of size and position of probes on signal intensity. Fragments of cDNA varying in size from 200 to 2000bp were generated by PCR using human IRF-1 cDNA as a template. Fragments were amplified either from the 3’ end (open circles) or from the 5’ end (squares) and immobilized onto glass slides in equal molarities. Synthetic RNA was prepared and labeled by RTase using oligo-dT as a primer. Average signal intensity from eight spots from each probe is represented as a function of probe size. Critical Reviews in Oncology / Hematology 2005 54, 95-105DOI: (10.1016/j.critrevonc.2004.12.006) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 The effect of probe position within the transcript. A glass array was printed with two distinct probes (clones Y and X) representing the same gene and co-hybridized with Cy3-labeled cDNA from a breast tumor sample and Cy5-labeled cDNA from immortalized breast luminal fibroblasts. For each gene, the expression ratio between the tumor and fibroblast samples for both clones was plotted in the log2 scale (a minus signal indicated higher expression in the fibroblast samples). In the lower panel, we depict the details of the two clones representing the gene indicated by the arrowhead. Critical Reviews in Oncology / Hematology 2005 54, 95-105DOI: (10.1016/j.critrevonc.2004.12.006) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Reproducibility of array data revealed by hierarchical clustering of replica hybridizations using the 4.8K universal chip made with ORESTES clones. A total of 37 samples representing normal and diseased samples from stomach and esophagus were hybridized in duplicate with dye-swap. Samples were clusterized according to their expression profile (correlation distance) considering all the spots with signal higher than background. In green we have samples representing normal tissue and in red we have samples representing diseased tissues. The red line and arrowheads indicate the single paired sample that did not clusterized at the first level. Critical Reviews in Oncology / Hematology 2005 54, 95-105DOI: (10.1016/j.critrevonc.2004.12.006) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Identification of intronic transcripts correlated to the degree of differentiation of prostate tumors. Hierarchical clustering of 66 partial cDNAs (38 protein-coding exons, 21 non-coding intronic transcripts and 7 intergenic ESTs) statistically correlated to Gleason score (p≤0.001). Intronic transcripts have their names in blue. The Gleason score (GS) of each sample is displayed at the bottom of the picture immediately after the patient number. The expression level of each gene (row) in each tumor sample (column) is represented by the number of standard deviations above (red) or below (blue) the average value for that gene across all the tumor samples. Critical Reviews in Oncology / Hematology 2005 54, 95-105DOI: (10.1016/j.critrevonc.2004.12.006) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions