Glass-slide microarrays

The result is high-quality arrays that can be used with a fluorescence detection system in a manner similar to the glass slide microarrays, although only single color detection is used. cDNA is synthesized from a test RNA sample by reverse transcription followed by second strand synthesis. This is then used as a template for an in vitro transcription reaction to produce biotin-labeled cRNA. As well as introducing the label for detection, this step permits amplification of the RNA and thus enables less starting material to be used. The cRNA is hybridized to the GeneChip and the biotin detected with an avidin-... 

DNA microarrays, or DNA chips consist of thousands of individual DNA sequences arrayed at a high density on a single matrix, usually glass slides or quartz wafers, but sometimes on nylon substrates. Probes with known identity are used to determine complementary binding, thus allowing the analysis of gene expression, DNA sequence variation or protein levels in a highly parallel format. 

The DNA microarrays are spotted on glass slides coated with amino-silane (Coming GAPS II). They should be ready for hybridization immediately when the labeled cDNA is ready. Thus, while the dyes are couplingto the cDNA (step 3 in the previous section), it is recommended to start the following process. 

Le Berre, V. Trevisiol, E. Dagkessamanskaia, A. Sokol, S. Caminade, A. M. Majoral, J. P. Meunier, B. Francois, J., Dendrimeric coating of glass slides for sensitive DNA microarrays analysis, Nucleic Acids Res. 2003, 31, e88... 

To use the microarray, a known sequence of short DNA is printed onto a solid support of membrane or glass slide. From healthy and diseased cells, mRNAs are isolated. The mRNAs are used to generate complementary DNAs (cDNAs). Fluorescent tags are attached to the cDNAs, and the cDNAs are then mixed and incubated with the microarray supports (slides). 

For example (a) Small-Molecule Microarrays Covalent Attachment and Screening of Alcohol-Containing Small Molecules on Glass Slides, P. ). Hergenr-OTHER, K. M. Depew, S. L. Schreiber,... 

Nitrocellulose and nylon membranes have been widely used in the production of macroarrays (arrays with probe sites of diameter 0.5 to 1 mm), but not so much in the production of microarrays (feature size of 25 to 200 xm) because of a lack of spot resolution (see Sect. 5.2, Spotting of Capture Probes). These membranes exhibit lateral wicking characteristics and the probe therefore tends to spread out from the point of apphcation. Casting of these membranes onto the surface of glass slides is a solution to this problem [28]. 

Membranes cast upon glass slides also fall into the 3D surface category. Most notable are the nitrocellulose-coated FAST slides offered by Schleicher Schuell (S S) BioScience. A cytokine micro-ELISA product xmder the trade name Provision has been introduced in single-slide and 96-well spacing (64 usable wells) formats. It is called FAST Quant (Harvey, 2003). Historically, nitrocellulose membrane has been used for the sequestering of both proteins and nucleic acids. The adaptation by S S of this microporous (0.2-p pores) material cast into a microarray format has been relatively straightforward. 

Both proteins and nucleic acids may be immobilized to a variety of solid supports. For high density microarrays, glass slides are the preferred substrates because of their flatness and optical properties. Better spot resolution is also possible on nonporous glass as opposed to porous membranes, primarily due to a reduction in diffusion at the surface-liquid interface. However, keep in mind that spot (droplet) diffusion can occur on most substrates by the actions of surfactants and other wetting agents including proteins. Control of spot size and morphology is required in order to achieve reproducible and reliable results with microarrays. 

Substrates for the creation of protein microarrays were initially selected from those used for DNA arrays, for example, PLL glass slides. At first, these substrates proved to be sufficient for antibody microarray studies. However, not all proteins will behave well or similarly on a particular substrate material. New solid phases applicable for protein microarrays need to be found. 

The primary substrate for spotted arrays is the glass slide. The salient physical and chemical features of a microarray slide are optical clarity (including... 

Perhaps the first published demonstration of high density applications for protein microarrays came from the work of MacBeath and Schreiber at Harvard (2000). Proteins were arrayed onto aldehyde-activated glass slides and analyzed in much the same manner described for the creation of cDNA microarrays (Schena et al., 1995), including the use of dual-color label... 

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