BACKGROUNDAffymetrix microarrays have become increasingly popular in gene-expression studies; however, limitations of the technology have not been well established for commercially available arrays. The hybridization signal has been shown to be proportional to actual transcript concentration for specialized arrays containing hundreds of distinct probe pairs per gene. Additionally, the technology has been described as capable of distinguishing concentration levels within a factor of 2, and of detecting transcript frequencies as low as 1 in 2,000,000. Using commercially available arrays, we assessed these representations directly through a series of 'spike-in' hybridizations involving four prokaryotic transcripts in the absence and presence of fixed eukaryotic background. The contribution of probe-target interactions to the mismatch signal was quantified under various analyte concentrations.RESULTSA linear relationship between transcript abundance and signal was consistently observed between 1 pM and 10 pM transcripts. The signal ceased to be linear above the 10 pM level and commenced saturating around the 100 pM level. The 0.1 pM transcripts were virtually undetectable in the presence of eukaryotic background. Our measurements show that preponderance of the signal for mismatch probes derives from interactions with the target transcripts.CONCLUSIONSLandmark studies outlining an observed linear relationship between signal and transcript concentration were carried out under highly specialized conditions and may not extend to commercially available arrays under routine operating conditions. Additionally, alternative metrics that are not based on the difference in the signal of members of a probe pair may further improve the quantitative utility of the Affymetrix GeneChip array.