Methodology article

Novel design and controls for focused DNA microarrays: applications in quality assurance/control and normalization for the Health Canada ToxArray™

Carole L Yauk¹ , Andrew Williams² , Sherri Boucher¹ , Lynn M Berndt¹ , Gu Zhou¹ , Jenny L Zheng¹ , Andrea Rowan-Carroll¹ , Hongyan Dong¹ , Iain B Lambert³ , George R Douglas¹ and Craig L Parfett¹

¹  Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Program, Health Canada, Ottawa, Ontario, K1A 0L2, Canada

²  Biostatistics and Epidemiology Division, Safe Environments Program, Health Canada, Ottawa, ON, K1A 0K9, Canada

³  Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.

author email corresponding author email

BMC Genomics 2006, 7:266doi:10.1186/1471-2164-7-266

Published:	19 October 2006

Abstract

Background

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. 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.

Results

An 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.

Conclusion

The 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.