The means of discovery of molecular signatures of response, long-term outcome, and subtype definition took a great leap forward with the advent of microarray technology (7,8). By allowing the simultaneous measurement of the mRNA expression of 100s to 1000s of genes from across the genome, array technology offered the potential to screen thousands of genes without the need for preconceived hypotheses.
Microarrays can investigate mitochondrial RNA (mRNA) or genomic deoxyribonucleic acid (DNA). Genomic DNA arrays can reveal large-scale gains and losses or mutations and polymorphisms, whereas mRNA targeted arrays allow gene-expression to be studied. An array is constructed on a solid substrate, often termed the "platform," made of either glass (microarray) or nitrocellulose or nylon (macroarrays). Two types of array dominate the expression profiling field: "chips" where oligonucleotide probes are grown out from the substrate surface, and "dot-blot" slides or membranes, where tiny dots of probe solution are blotted on to the substrate. A third type "oligonucleotide ink-jet/ piezo" arrays, has been developed more recently and will be described briefly. For a list of commercial suppliers of arrays, visit http://www.lab-on-a-chip.com and http://ihome.cuhk.edu.hk/~b400559/array.html.
On the substrate, 100s (macroarrays) to 1000s (microarrays) of complimentary DNA (cDNA) amplicons or oligonucleotides are immobilized in an ordered, grid pattern. Confusingly, the arrayed cDNA amplicons or oligonucleotides are called "targets" in most systems but "probes" in Affymetrix chips. Because this chapter refers to the use of the Affymetrix system, the cDNA amplicons or oligonucleotides will be referred to as "probes" henceforth. To the "probes,"
labeled complementary (c)RNA, cDNA, or genomic DNA are hybridized in a manner similar to that of Southern and Northern blotting. (In most systems, the labeled cRNA, cDNA, or genomic DNA is referred to as the "probe," whereas in the Affymetrix system, and the remainder of this chapter, it is referred to as the "target.") Each of the immobilized "probes" acts as an assay for its specific partner strand in the complex nucleic acid mixture of the target. The target labels can be fluorophores, biotin, or radioactive phosphorus. Arrays designed for radioactive labeled target (macroarrays) require as little as 50 ng RNA (5000 cells), compared with the minimum of 5 ^g (500,000 cells) required for fluorescent systems. 33P-based arrays, however, produce reliable readouts only for highly expressed genes, which are the minority. Fluorescence-dependent arrays allow the detection of low- and medium-abundance genes, which are the majority, as well as those genes highly expressed at the mRNA level. It is the fluorescence-dependent arrays that have been used to great effect in the study of DLBCL and that will now be described in more detail.
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