The beta test sites were provided probe arrays, four hybridization targets containing spiked transcripts (at four concentrations, discussed later in the document), staining reagents, a GeneChip Scanner 3000 with experimental instructions, and support from Affymetrix. Beta test participants were responsible for acquiring images on both the GeneChip Scanner 3000 and GeneArray 2500, returning the raw data to Affymetrix, and providing detailed feedback on the GeneChip Scanner 3000.
Hybridization targets (labeled and fragmented cRNA ready for hybridization) were prepared from human cell line total RNA following procedures outlined in the GeneChip Expression Analysis Technical Manual.
Assay performance was evaluated, in part, on the ability of each scanner type to detect and quantify 53 labeled cRNA transcripts spiked into the target sample. These 53 transcripts were selected on the basis of their apparent lack of expression in the source RNA, as determined by their Absent calls on GeneChip human genome probe arrays.
The spiked transcripts were generated by in vitro transcription from vectors containing the cloned DNA sequence. Labeled cRNA transcripts were then quantified and fragmented and spiked back into labeled, fragmented, complex background cRNA, to reconstitute expression of all 53 transcripts at one of three concentrations: 0.75 pM (picomolar), 1.50 pM, or 3.00 pM. A target sample created from background cRNA without spiked tra