title: Novel design and controls for focused DNA microarrays: applications in quality assurance/control and normalization for the Health Canada ToxArray.
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 microM) 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Program, Health Canada, Ottawa, Ontario, K1A 0L2, Canada. carole_yauk@hc-sc.gc.ca
authors.forename: Carole L
authors.initials: CL
authors.lastname: Yauk
authors.forename: Andrew
authors.initials: A
authors.lastname: Williams
authors.forename: Sherri
authors.initials: S
authors.lastname: Boucher
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Berndt
authors.forename: Gu
authors.initials: G
authors.lastname: Zhou
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Zheng
authors.forename: Andrea
authors.initials: A
authors.lastname: Rowan-Carroll
authors.forename: Hongyan
authors.initials: H
authors.lastname: Dong
authors.forename: Iain B
authors.initials: IB
authors.lastname: Lambert
authors.forename: George R
authors.initials: GR
authors.lastname: Douglas
authors.forename: Craig L
authors.initials: CL
authors.lastname: Parfett
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D016477:Artifacts; D002170:Canada; D005188:False Negative Reactions; D005189:False Positive Reactions; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D011786:Quality Control; D018075:RNA, Complementary / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018075:RNA, Complementary; D012333:RNA, Messenger