title: Assessing probe-specific dye and slide biases in two-color microarray data.
abstract: BACKGROUND
  A primary reason for using two-color microarrays is that the use of two samples labeled with different dyes on the same slide, that bind to probes on the same spot, is supposed to adjust for many factors that introduce noise and errors into the analysis. Most users assume that any differences between the dyes can be adjusted out by standard methods of normalization, so that measures such as log ratios on the same slide are reliable measures of comparative expression. However, even after the normalization, there are still probe specific dye and slide variation among the data. We define a method to quantify the amount of the dye-by-probe and slide-by-probe interaction. This serves as a diagnostic, both visual and numeric, of the existence of probe-specific dye bias. We show how this improved the performance of two-color array analysis for arrays for genomic analysis of biological samples ranging from rice to human tissue.
  
  
  RESULTS
  We develop a procedure for quantifying the extent of probe-specific dye and slide bias in two-color microarrays. The primary output is a graphical diagnostic of the extent of the bias which called ECDF (Empirical Cumulative Distribution Function), though numerical results are also obtained.
  
  
  CONCLUSIONS
  We show that the dye and slide biases were high for human and rice genomic arrays in two gene expression facilities, even after the standard intensity-based normalization, and describe how this diagnostic allowed the problems causing the probe-specific bias to be addressed, and resulted in important improvements in performance. The R package LMGene which contains the method described in this paper has been available to download from Bioconductor.
authors.affiliation: Department of Data Analysis and Algorithm, Affymetrix, Inc., Santa Clara, California, USA. tinypenguin@gmail.com
authors.forename: Ruixiao
authors.initials: R
authors.lastname: Lu
authors.forename: Geun-Cheol
authors.initials: GC
authors.lastname: Lee
authors.forename: Michael
authors.initials: M
authors.lastname: Shultz
authors.forename: Chris
authors.initials: C
authors.lastname: Dardick
authors.forename: Kihong
authors.initials: K
authors.lastname: Jung
authors.forename: Jirapa
authors.initials: J
authors.lastname: Phetsom
authors.forename: Yi
authors.initials: Y
authors.lastname: Jia
authors.forename: Robert H
authors.initials: RH
authors.lastname: Rice
authors.forename: Zelanna
authors.initials: Z
authors.lastname: Goldberg
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Schnable
authors.forename: Pamela
authors.initials: P
authors.lastname: Ronald
authors.forename: David M
authors.initials: DM
authors.lastname: Rocke
mesh_terms: D000465:Algorithms; D015342:DNA Probes / Q000235:genetics*; D005456:Fluorescent Dyes / Q000032:analysis*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015588:Observer Variation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes