Currently the solid supports upon which nucleic acids are arrayed are either glass slides or nylon membranes. Typically, fluorescently labeled probes are used with glass arrays, while radiolabeled probes are used with membranes. Depending on the type of array, the arrayed nucleic acids may be composed of oligonucleotides, PCR products or cDNA vectors or purified inserts. The sequences may represent entire genomes and may include both known and unknown sequences or may be collections of sequences such as apoptosis-related genes or cytokines. Many pre-made and custom arrays are available from commercial manufacturers although many labs prepare their own arrays with the help of robotic arrayers. The methods of probe labeling, hybridization, and detection depend on the solid support to which the sequences are bound.
Many terms exist for naming gene arrays, including biochip, DNA chip, GeneChip (a registered trademark of Affymetrix, Inc.), DNA array, microarray and macroarray. Generally when biochip, DNA chip or GeneChip is used, it refers to arrays on glass supports. Microarray and macroarray may be used to differentiate between spot size or the number of spots on the support. Gene arrays may be used for sequence identification (e.g. mutation analysis) or differential expression analysis of two or more RNA samples. This discussion will focus on the use of arrays for expression analysis.
Some array manufacturers offer custom analysis services and may perform the probe labeling and hybridization reactions as a service.
Nylon Membrane Arrays
Nylon membrane arrays are typically hybridized with 33P-dNTP labeled probes and analyzed by a phosphorimager along with the appropriate software. A different array must be used for each sample analyzed. A typical experiment involves isolating RNA from two tissue or cell samples. The RNAs are reverse transcribed using labeled nucleotides and target specific, oligo dT, or random-sequence primers to create two labeled cDNA populations. The two cDNAs are hybridized to two identical arrays. After washing, the hybridized signal on each array is detected and analyzed. The signal emitting from each gene-specific spot is compared between the populations. Genes expressed at different levels in two samples generate different amounts of labeled cDNA and this results in spots on the array with different amounts of signal.
Glass Slide Arrays
Glass slide arrays analysis involves the same steps, but rather than labeling with isotopes during reverse transcription, probes for glass arrays are labeled with two distinct fluorescently labeled nucleotides and both probes are hybridized to the same array. Typically, one sample RNA is labeled with Cyanine 3-dNTP (Cy3) and the other with Cyanine 5-dNTP (Cy5). Each dye produces different color fluorescence. The two labeled RNA populations are hybridized to one glass slide and scanned using a fluorescent imager.
Affymetrix's GeneChips are glass slide arrays manufactured using special photolithographic methods and combinatorial chemistry, which allow the oligonucleotide spots to be synthesized directly onto the array substrate. The analysis procedure specifies that the RNA samples are converted to biotin-labeled cDNA, and each sample is hybridized to a separate GeneChip. The hybridized cDNA is then stained with a streptavidin-phycoerythrin conjugate and visualized with an array scanner.
Where once the bottleneck in gene expression analysis was the benchwork, with array analysis, it is the computer work. Because a single array experiment can generate thousands of data points, the primary challenge of the technique is making sense of the data. Many commercial companies provide image analysis software, including BioDiscovery (ImaGene) and Imaging Research (ArrayVision). Furthermore, many array manufacturers offer software specifically for the analysis of their arrays and offer the analysis as a service.
For membrane array analysis, a file of the data is generated by phosphoimaging and that file is then analyzed using software. The software will correlate spots to genes and can compare spot intensities for differential expression studies.
Glass array data is treated in much the same way, but the image's fluorescence is scanned and the software allows detection of each samples' fluorescence individually or simultaneously for analysis. Most software packages can analyze several arrays simultaneously.
Differences in expression of specific sequences are often validated by another method of analysis such as RT-PCR, Northern analysis or nuclease protection assays. These same methods can be used for relative or absolute quantitation of specific messages of interest identified by array analysis.
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