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Related Experiment Videos

Pre-processing Agilent microarray data.

Marianna Zahurak1, Giovanni Parmigiani, Wayne Yu

  • 1Johns Hopkins University School of Medicine, Oncology Biostatistics, Baltimore, MD 21205, USA. zahurma@jhmi.edu

BMC Bioinformatics
|May 3, 2007
PubMed
Summary
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Simple loess normalization without background subtraction offers reliable gene expression ranking for Agilent arrays. This method, while biasing fold changes, improves detection of differentially expressed genes compared to standard Agilent processing.

Area of Science:

  • Genomics
  • Bioinformatics
  • Microarray Analysis

Background:

  • Pre-processing methods for Agilent oligonucleotide arrays are not well-established.
  • Sources of error in Agilent array expression measurement require quantification.
  • Comparison of Agilent Feature Extraction software with standard cDNA array normalization is needed.

Purpose of the Study:

  • To quantify error sources in Agilent array expression measurement.
  • To compare Agilent Feature Extraction with standard cDNA array normalization methods.
  • To define optimal study design and pre-processing practices for Agilent arrays.

Main Methods:

  • Log transformation, loess normalization (with/without background subtraction), and between-array scaling were compared.
  • Receiver Operating Characteristic (ROC) analysis was used on a spike-in experiment.

Related Experiment Videos

  • Dye effects were assessed across different pre-processing methods.

    • Main Results:

    • Loess normalization without background subtraction yielded the lowest variability and highest AUC (99.7%) for detecting differentially expressed genes.
    • Without background subtraction, fold changes were biased towards zero at low intensities.
    • 43% of genes showed dye effects, though generally small across methods.

    Conclusions:

    • Simple loess normalization without background subtraction provides reliable fold changes for gene expression ranking.
    • While statistics like t- or z-statistics are robust, fold changes remain crucial for exploratory analysis and interpretation.
    • Experimental designs incorporating dye swaps or common references are recommended due to prevalent dye effects.