Affymetrix slides

The second approach for microarray analyses is based on Affymetrix slides and consists of hundreds to thousands of sets of 12-25 pairs of short oligos synthesized... 

Oligonucleotide arrays [6] In-situ on the surface of the matrix 1 cm by 1 cm slide with approximately 40 000 genes Affymetrix s GeneChip can contain up to 400 000 different oligonucleotides and is the densest array Fluorescent tag labeling fluorophore detector is added after hybridization Passive Affymetrix... 

Diluted human serum (1 pL) was incubated with the peptide microarray and bound antibodies were detected using a rhodamine-labeled anti-human IgG. Signal was detected using a slide scanner (Affymetrix model 418) with data collection in the Cy3 channel. A reported eightfold gain in sensitivify at 100% specificity over standard ELISA was achieved using the peptide microarray. 

For Affymetrix (Santa Clara, CA) (see below) and for other single-channel experiments, the hybridization scheme is straightforward, but for two-channel platforms, the allocation of samples to arrays is important to prioritize the primary scientific question of the study. Furthermore, balanced designs should be used so that treatments are not confounded with technical issues such as dye assignments, batch of slides, or day of hybridization. Replication is carried out to control the three levels of variation in an experiment biologic variation (e.g., differences... 

Once the RNA is prepared, it is converted to cDNA and labeled either with radioactive precursors ([33P]-dCTP or [32P]-dCTP) or with non-radioactive fluorescent labels such as Cy3-dNTP and Cy5-dNTP. Radioactive labels are commonly used for membrane approaches while fluorescent probes are the method of choice for glass slides. When using fluorescent probes, cDNA prepared from controls can be labeled with one dye while cDNA from treated samples can be labeled with the other. These are then mixed and used in competitive binding to probes on the chip. Alternatively, one can compare all control and experimental samples to the same reference RNA sample, labeled with one of the dyes. Reference RNA samples are useful for multiple sample analysis and to account for chip-to-chip variations. In the case of the Affymetrix technique, cDNAs are converted back into fluorescently labeled RNA targets, which bind more tightly than cDNA targets to the short oligonucleotides present on those chips, and the control and experimental samples are applied individually to separate chips. 

Figure 2.2 Microarray slides Photo courtesy of Affymetrix. Inc., US.

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