Each Affymetrix GeneChip® probe set contains 8 to 16 paired perfect match (PM) and mismatch (MM) 25-mer probes, which are used to determine whether a given gene is expressed and to measure the expression level (signal) [4]. The Affymetrix Microarray Suite version 5 (MAS5) algorithm uses the probe-pair data in different ways to calculate the detection call and the signal. MAS5 uses a non-parametric statistical test (Wilcoxon signed rank test) of whether significantly more perfect matches show more hybridization signal than their corresponding mismatches to produce the detection call (Absent (A), Present (P) or Marginal (M)) for each probe set [5]. We will use the convention of capitalized Present, Absent, and Marginal to indicate the formal detection calls. The signal is the anti-log of an average (Tukey biweight) of the log(PM-MMadj) for all of the probe pairs, where MMadj is equal to MM or an adjusted quantity which will produce a positive value [6]. Genes that are not detectably expressed nevertheless generate signal values, usually low; random fluctuations in these low values can often produce large apparent fold-changes.
