A revised design for microarray experiments to account for experimental noise and uncertainty of probe response

Alex E. Pozhitkov, Peter A. Noble, Jarosław Bryk, Diethard Tautz

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Background: Although microarrays are analysis tools in biomedical research, they are known to yield noisy output that usually requires experimental confirmation. To tackle this problem, many studies have developed rules for optimizing probe design and devised complex statistical tools to analyze the output. However, less emphasis has been placed on systematically identifying the noise component as part of the experimental procedure. One source of noise is the variance in probe binding, which can be assessed by replicating array probes. The second source is poor probe performance, which can be assessed by calibrating the array based on a dilution series of target molecules. Using model experiments for copy number variation and gene expression measurements, we investigate here a revised design for microarray experiments that addresses both of these sources of variance. Results: Two custom arrays were used to evaluate the revised design: one based on 25 mer probes from an Affymetrix design and the other based on 60 mer probes from an Agilent design. To assess experimental variance in probe binding, all probes were replicated ten times. To assess probe performance, the probes were calibrated using a dilution series of target molecules and the signal response was fitted to an adsorption model. We found that significant variance of the signal could be controlled by averaging across probes and removing probes that are nonresponsive or poorly responsive in the calibration experiment. Taking this into account, one can obtain a more reliable signal with the added option of obtaining absolute rather than relative measurements. Conclusion: The assessment of technical variance within the experiments, combined with the calibration of probes allows to remove poorly responding probes and yields more reliable signals for the remaining ones. Once an array is properly calibrated, absolute quantification of signals becomes straight forward, alleviating the need for normalization and reference hybridizations.

Original languageEnglish
Article numbere91295
Number of pages10
JournalPLoS One
Volume9
Issue number3
DOIs
Publication statusPublished - 11 Mar 2014
Externally publishedYes

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Microarrays
probes (equipment)
Calibration
Uncertainty
Noise
uncertainty
Microarray Analysis
Adsorption
Biomedical Research
Experiments
Gene Expression
Dilution
calibration
Molecules
biomedical research
Gene expression
adsorption
hybridization

Cite this

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A revised design for microarray experiments to account for experimental noise and uncertainty of probe response. / Pozhitkov, Alex E.; Noble, Peter A.; Bryk, Jarosław; Tautz, Diethard.

In: PLoS One, Vol. 9, No. 3, e91295, 11.03.2014.

Research output: Contribution to journalArticle

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