Expression array

Biological raw data are stored in public databanks (such as Genbank or EMBL for primary DNA sequences). The data can be submitted and accessed via the World Wide Web. Protein sequence databanks like trEMBL provide the most likely translation of all coding sequences in the EMBL databank. Sequence data are prominent, but also other data are stored, e.g.yeast two-hybrid screens, expression arrays, systematic gene-knock-out experiments, and metabolic pathways. 

Applied Biosystems Expression Array System (Foster City, CA) has devised a chemiluminescence-based microarray platform ntilizing 60-mer oligonncleotides which are validated offline by mass spectrometry and are snbseqnently printed onto a derivatized nylon substrate. Agilent Technologies (Palo Alto, CA) also ntilizes 60-mers, which are synthesized in sitn by ink-jet printing nsing phosphoramidite chemistry. 

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

Antibody coverage of the human proteome is estimated to be about 5 to 10% of all human proteins and isoforms (Valle and Jendoubi, 2003). A major bottleneck in the use of protein expression arrays is the lack of such a comprehensive set of these capture agents (Hanash, 2003). Since an equivalent of the polymerase chain reaction (PCR) process for mass amplification of low abxmdant proteins does not exist, the remaining library of proteome capture ligands will need to be generated by other means such as recombinant protein expression systems (Cahill, 2001). 

Hornberg, J.J., de Hass, R.R., Dekker, H., and Lankelma, J., Analysis of multiple gene expression array experiments after repetitive hybridizations on nylon membranes, Biotechniques, 33, 108-117, 2002. 

This chapter will review some of the key applications presented by protein microarrays. The use of profein microarrays sfems from works on gene expression arrays described earlier. However, rmlike ifs predecessors whose process formats (mutation detection, polymorphism screening, gene expression analysis, etc.) are essentially based upon solid-phase hybridization of nucleic acid complementary strands, the protein array may play different roles and comprise a variety of formafs. 

Walker BA, Leone PE, Jermer MW et al. Integration of global SNP-based mapping and expression arrays reveals key regions, meehanisms, and genes important in the pathogenesis of multiple myeloma. P/oori 2006 108 1733-1743. 

Shim, C., Zhang, W., Ree, C. H., and Lee, J.-H. 1998. Profiling of differentially expressed genes in human primary cervical cancer by complementary DNA expression array. Clin. Cancer Res. 4 3045-3050. 

Fig. 4. Expression profile of cytokine genes in human lymphocytes detected by cDNA expression array system. RNA was extracted from human peripheral blood lymphocytes from (a) normal control subjects and (b) allergic asthmatic patients. Total RNA was reverse-transcribed and labeled with biotin, and gene expressions were detected using the Human Chemokine Nonrad-GEArray Kit (SuperArray Inc., Bethesda MD, USA). Negative control genes pUC18 DNA (1A, 2A) positive control genes /J-actin (3A, 4A) and GADPH (8B, 8C, 5A, 6A, 7A, 8A).

L3. Lapteva, N., Ando, Y., Nieda, M., Hohjoh, H., Okai, M., Kikuchi, A., Dymshits, G., Ishikawa, Y., Juji, T., and Tokunaga, K., Profihng of genes expressed in human monocytes and monocyte-derived dendritic cells using cDNA expression array. Br. J. Haematol. 114, 191-197 (2001). L4. Lauw, F. N., Dekkers, P. E., te Velde, A. A., Speelman, P., Levi, M., Kurimoto, M., Hack, C. E., van Deventer, S. J., and van der Poll, T., Interleukin-12 induces sustained activation of multiple host inflammatory mediator systems in chimpanzees. J. Infect. Dis. 179,646-652 (1999). 

Rae JM, Johnson MD, Lippman ME, Flockhart DA. Rifampin is a selective, pleiotropic inducer of drag metabolism genes in human hepatocytes studies with cDNA and oligonucleotide expression arrays. J Pharmacol Exp Ther 2001 299 849-857. 

Rocke DM, Durbin B. A model for measurement error for gene expression arrays. J Comput Biol 2001 8 557-569. 

There are some important limitations to the expression array approach. Since this analysis examines only the transcriptome, it fails to identify the important regulatory points at the level of translation and enzyme activity [29], in addition to the processes of protein protein interaction. Currently, the expression arrays do not address the issue of alternative splicing, but there are new generations of arrays being produced to examine these events. 

It is not possible to review all applications of gene expression arrays in the field of cancer and a number of excellent reviews are available to examine different aspects of the field, for example [43-48]. However, as an introduction to how the methodology has been applied, we will discuss breast cancer as an illustrative example. 

Galamb O, Sipos F, Fischer K, Tulassay Z, Molnar B. The results of the expression array studies correlate and enhance the known genetic basis of gastric and colorectal cancer. Cytometry B Clin Cytom 2005 68(1) 1—17. 

---