Overview of the production and use of expression microarrays. 3′ Expression arrays use synthetically derived oligo probes with design based on mRNA Databases (RefSeq mRNAs, GenBank mRNAs, and ESTs from dbEST) or cDNA derived from bacterial libraries (see Figure 1). Sample mRNA can be labeled using two methods (a) Cy3/Cy5 labeling: sample mRNA is reverse transcribed into cDNA and Cy3 is added to one sample and Cy5 to another. Both labeled samples are hybridized to the same microarray. (b) 3′ IVT array: sample mRNA is reverse transcribed to cDNA using oligo(dT) primers, to provide a template for transcription. Using biotin-conjugated nucleotides, the template cDNA is then converted to amplified RNA (aRNA). The biotin-labeled aRNA samples are then fragmented and hybridized onto 3′ expression arrays. A biotin binding fluorescent stain is added to the microarray after hybridization. (c) Affymetrix HuExon 1.0 ST: sample mRNA is reverse transcribed to cDNA using random primers, to provide a template for transcription. The resulting RNA is then reverse transcribed in the presence of dUTPs which are incorporated occasionally into the cDNA sequence instead of dTTP. An enzyme is then used to cleave the cDNA at the site of dUTP incorporation and fragments are terminally labeled before hybridization onto the array. The microarray is then washed and stained after hybridization.
