Figure 2

Functional genomics of MS. (a) Accurate diagnosis of patients based on clinical evidence, particularly magnetic resonance imaging, and the patients' historical data, is pivotal for the validity of genomic analysis. (b) Typically, post-mortem samples with short autolysis times are used, although biopsy specimens are sometimes available. Immunohistological analysis and laser capture microdissection at this stage can add substantially to subsequent data interpretation. (c) High-quality RNA is then obtained from the tissue and subjected to quantitation and integrity analysis. (d) Depending on the type of array to be used, the RNA sample is converted into fluorophore-labeled cDNA (spotted cDNA arrays) or cRNA (oligonucleotide-based arrays). (e) A nucleic-acid-containing solid support - the DNA chip or microarray - is obtained either by spotting cDNA clones or by in situ synthesis of oligonucleotides onto a glass surface. (f) The labeled sample is then laid on top of the array and hybridized for several hours. In the case of spotted cDNA arrays, an equal amount of two differently labeled samples (usually one is a control) is mixed prior to the hybridization step. (g) A confocal laser microscope can be used to scan and measure the fluorescence emitted by the hybridized probes. The intensity of the signal is directly related to the amount of mRNA originally present in that sample. In spotted cDNA arrays, the ratio of the two fluorophores is measured and the relative intensity of each probe is then calculated for each cDNA-containing spot. (h) Different classification algorithms can be used to organize the expression of all genes analyzed in a particular experiment. In this way, genes with correlated patterns of expression are clustered together and so can be readily identified. (i) On the basis of the expression results, a particular gene or group of genes can be selected for validation in vivo. At this stage, animal models can be used to assess the effect of a genetic deletion or overexpression affecting the gene(s) of interest. (j) At the end of this process, a hypothesis can be generated that is consistent with the results obtained. New rounds of experimentation are usually required to refine a particular hypothesis.