Abstract
BackgroundMicroscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs).ResultsWe present a method that harnesses the statistical power provided by having several HDONAs available, which are obtained under similar conditions except for the experimental factor. This method "harshlights" blemishes and renders them evident. We find empirically that about 25% of our chips are blemished, and we analyze the impact of masking them on screening for differentially expressed genes.ConclusionExperiments attempting to assess subtle expression changes should be carefully screened for blemishes on the chips. The proposed method provides investigators with a novel robust approach to improve the sensitivity of microarray analyses. By utilizing topological information to identify and mask blemishes prior to model based analyses, the method prevents artefacts from confounding the process of background correction, normalization, and summarization.
Highlights
Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc
To study how the immune response of leukocytes isolated from blood can be affected by drugs that may serve to control autoimmune diseases like psoriasis, blood was drawn from five volunteers under a protocol that had been approved by The Rockefeller University Hospital Institutional Review Board [12]
One of the two sets of control, activated, and pre-treated cultures was analyzed after 6, the other after 24 hrs. (For subjects 3, 4, and 5, only one time point is available.) All samples were hybridized to Affymetrix HuU95av2 chips
Summary
Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Most blemishes are hard to find by the unaided eye, in high-density oligonucleotide arrays (HDONAs). Analysis of hybridized microarrays starts with scanning the fluorescent image. For high-density oligonucleotide arrays (HDONAs) such as Affymetrix GeneChip® oligonucleotide (Affy) arrays, the focus of this paper, each scanned image is stored pixel-by-pixel in a 'DAT' file. As the first step in measuring intensity of the hybridization signal, a grid is overlaid, the image is segmented into spots or features, and the pixel intensities within each of these are summarized as a probe intensity estimate (See reviews [1] and [2] for cDNA chips). Expression of a gene is estimated from such a probe set by applying algorithms for background correction, normalization, and summarization
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