The gene expression assaying technologies that will be discussed in this chapter are RNA detection microarrays, variously known as DNA chips, biochips, or simply chips. It is easiest to think of the utility of microarrays in functional genomics as a five-step process.
• Probe: This is the biochemical agent that finds or complements a specific sequence of DNA, RNA, or protein from a test sample . It can include pieces of cDNA amplified from a vector stored within a bacterial clone, oligonucleotides synthesized in a fluid medium, oligonucleotides built on a solid phase base pair by base pair, or nucleotide fragments of a chromosome. This can be extended to incorporate the database of expressed sequence tags (ESTs) for serial analysis of gene expression (SAGE), (see Glossary, page 277).
• Array: The method for placing the probes on a medium or platform. Current techniques include robotic spotting, electric guidance, photolithography, piezoelectricity, fiber optics, and microbeads. This step also specifies the type of medium involved, such as glass slides, nylon meshes, silicon, nitrocellulose, membranes, gels, and beads.
• Sample probe: The mechanism for preparing RNA from test samples. Total RNA may be used, or mRNA may be selected using a polydeoxythymidine (poly-dT) to bind the polyadenine (poly-A) tail. Alternatively, mRNA may be copied into cDNA, using labeled nucleotides or biotinylated nucleotides.
• Assay: How is the signal of gene expression being transduced into something more easily measurable? For the microarrays in common use, gene expression is transduced into hybridization. For SAGE, gene expression is transduced into oligonucleotides via restriction enzymes and ligation. For PCR, gene expression is transduced into amplified pieces of cDNA.
• Readout: How is the transduced signal going to be measured? How is information about the signal represented? For the microarrays in common use, hybridization is typically measured either using one or two colored dyes, or radioactive labels. For SAGE, the constructed oligonucleotides are measured through sequencing. For PCR, the amplified pieces of cDNA can be measured using gel electrophoresis.
For the microarrays in common use, one typically starts by taking a specific biological tissue or system of interest, extracting its mRNA, and making a fluorescence-tagged cDNA copy of this mRNA. This tagged cDNA copy, typically called the sample probe, is then hybridized to a slide containing a grid or array of single-stranded cDNAs called probes which have been built or placed in specific locations on this grid (see table 3.1 for alternative terminology). Similar to the general hybridization principles behind Southern or Northern blots (see glossary, page 277), a sample probe will only hybridize with its complementary probe. Fluorescence is typically added to the sample probe in one of two ways: Either (1) fluorescent nucleotide bases are used when making the cDNA copy of the RNA, or (2) biotinylated nucleotides are first incorporated, followed by an application of fluorescence-labeled streptavidin, which will bind to the biotin. Depending upon manufacturer-specific protocols, the probe-sample probe hybridization process on a microarray typically occurs over several hours. All unhybridized sample probes are then washed off and the microarray is lit under laser light and scanned using laser confocal microscopy analogous to the phosphor imager in the traditional blot procedures. A digital image scanner records the brightness level at each grid location on the microarray corresponding to particular RNA species.
Table 3.1: Common probe nomenclature
Immobilized nucleic acid on microarray surface
Free nucleic acid that is being interrogated
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