Abstract
BackgroundMicroarray normalizations typically apply methods that assume absence of global transcript shifts, or absence of changes in internal control features such as housekeeping genes. These normalization approaches are not appropriate for focused arrays with small sets of genes where a large portion may be expected to change. Furthermore, many microarrays lack control features that can be used for quality assurance (QA). Here, we describe a novel external control series integrated with a design feature that addresses the above issues.ResultsAn EC dilution series that involves spike-in of a single concentration of the A. thaliana chlorophyll synthase gene to hybridize against spotted dilutions (0.000015 to 100 μM) of a single complimentary oligonucleotide representing the gene was developed. The EC series is printed in duplicate within each subgrid of the microarray and covers the full range of signal intensities from background to saturation. The design and placement of the series allows for QA examination of frequently encountered problems in hybridization (e.g., uneven hybridizations) and printing (e.g., cross-spot contamination). Additionally, we demonstrate that the series can be integrated with a LOWESS normalization to improve the detection of differential gene expression (improved sensitivity and predictivity) over LOWESS normalization on its own.ConclusionThe quality of microarray experiments and the normalization methods used affect the ability to measure accurate changes in gene expression. Novel methods are required for normalization of small focused microarrays, and for incorporating measures of performance and quality. We demonstrate that dilution of oligonucleotides on the microarray itself provides an innovative approach allowing the full dynamic range of the scanner to be covered with a single gene spike-in. The dilution series can be used in a composite normalization to improve detection of differential gene expression and to provide quality control measures.
Highlights
Microarray normalizations typically apply methods that assume absence of global transcript shifts, or absence of changes in internal control features such as housekeeping genes
Description and positioning of control spots We developed an external control (EC) dilution series using the Arabidopsis thaliana chlorophyll synthase gene that involves spike-in of a single concentration of the chlorophyll synthase cRNA to hybridize against spotted dilutions (0.000015 to 100 μM) of a single oligonucleotide representing the gene within each sub-grid of the ToxArrayTM (Figure 1 and 2; see Methods)
We developed an EC dilution series using the spike-in of a single Arabidopsis chlorophyll synthase gene hybridized to a series of dilutions of the complementary oligonucleotide printed on the microarray
Summary
Microarray normalizations typically apply methods that assume absence of global transcript shifts, or absence of changes in internal control features such as housekeeping genes. These normalization approaches are not appropriate for focused arrays with small sets of genes where a large portion may be expected to change. High-density genomic tools, such as DNA microarrays, provide an important opportunity to study the global response of genomes to particular stressors or conditions. Focused arrays that contain a limited number of features associated with particular biochemical pathways often lack technical and biological control features, such as gene replicates, quality assurance controls and/or normalization features, that are required for experiments in which a large proportion of genes may exhibit differential expression. The use of custom-made focused microarrays has become a popular alternative, allowing the study of genes relevant to a specific hypothesis
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