Gene profiling--chances and challenges.

Abstract

Microarray analysis has been emerged as a tool to characterize the overall reaction of cells in culture or tissue to different stimuli e.g. stressful events by analysing bulk RNA present at a particular time point. It has supplemented or even replaced more traditional methods like cDNA-bank sequencing or conventional differential display. The commercial availability of several different precoated arrays and the ease of handling has supported the broad distribution of this new technique. The basic protocol involves the hybridization of complementary strands of labelled DNA or RNA from cells/tissue with representations of known genes spotted onto a solid support (nylon, glass). Labelling can be radioactive (p32/33), by a hapten group (biotin, digoxigenin, aminoallyl) or by fluorescent (Cy3, Cy5 etc.) nucleotides. Detection is performed by autoradiography, chemiluminescence or fluorescence scanning. There are different setups of arrays available: either known genes/gene-groups (apoptosis, cytokines etc.) are spotted as PCR fragments, plasmids or synthetic oligonucleotides or representations of the known genome are directly synthesized as short sequence tags of 20-70 oligonucleotides on glass chips. The latter allow the identification of newly expressed genes whereas the former deal with known genes. Ideally, the intensity of the signal can be correlated with the relative expression of a known gene and allows the comparison with a standard. Problems arise from the quality of the sample material, the standardization of the protocols and the data management. Nevertheless, gene profiling by cDNA-arrays will definitely be integrated into routine screening programs.