Preferred analysis methods for Affymetrix GeneChips. II. An expanded, balanced, wholly-defined spike-in dataset

We present a new wholly defined Affymetrix spike-in dataset consisting of 18 microarrays. Over 5700 RNAs are spiked in at relative concentrations ranging from 1- to 4-fold, and the arrays from each condition are balanced with respect to both total RNA amount and degree of positive- versus negative-fold change. We use this new “Platinum Spike” dataset to evaluate microarray analysis routes and contrast the results to those achieved using our earlier Golden Spike dataset. PCR products from 5725 Drosophila Gene Collection release 1.0 (DGCr1) cDNA clones were collected into 28 distinct pools, and three independent in vitro transcription and labeling reactions were performed for each pool. Labeled cRNAs from each individual pool were then added at specified amounts to samples A and B to achieve the desired fold change differences between samples. 24 cRNAs generated from DGCr2 cDNA clones were added to each sample in known concentrations before hybridization.