Research

Assessment of the relationship between signal intensities and transcript concentration for Affymetrix GeneChip^® arrays

Eugene Chudin^1,4 , Randal Walker^2,4, Alan Kosaka^2,4, Sue X Wu^2,4, Douglas Rabert², Thomas K Chang² and Dirk E Kreder³

¹  Illumina, San Diego, CA 92129, USA

²  Roche Bioscience, Palo Alto, CA 94304, USA

³  Abgenix Inc., Fremont, CA 94555, USA

⁴  These authors contributed equally to this work

author email corresponding author email

Genome Biology 2001, 3:research0005.1-0005.10doi:10.1186/gb-2001-3-1-research0005

Published:	14 December 2001

Subject areas: Bioinformatics, Methods, Genome studies

Abstract

Background

Affymetrix 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.

Results

A 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.

Conclusions

Landmark 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.