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Quality assessment of microarray data in a multicenter study.

ABSTRACT: Issues implicit in a multicenter microarray study are protocol standardization and monitoring center adherence to established protocols. This study explored the effects of submitting center and sample preservation method on the quality of isolated RNA. In addition, the effects of sample preservation method and laboratory on microarray quality were also examined. Herein we evaluated the contribution of specific technical factors [center, laboratory, and preservation method (frozen/RNAlater)] on quality of isolated RNA, cRNA synthesis products, and reproducibility of gene expression microarray data for independent biologic samples collected in a multicenter microarray study. The Kruskal-Wallis test was used to test for differences owing to submitting center on isolated RNA quality. Mixed effects analysis of variance was used in assessing the impact of laboratory and preservation method on gene expression values for the 12 samples hybridized at 2 independent laboratories (24 GeneChips). One center was found to be in violation of the tissue handling protocol. No significant effect was noted owing to preservation method, which ensured that our tissue handling protocols are working properly. There was a significant laboratory effect with respect to cRNA yield, though this effect did not impact sample quality. We conclude that use of consistent protocols for sample collection, RNA extraction, cDNA/cRNA synthesis, labeling, hybridization, platform, image acquisition, normalization, and expression summaries can yield consistent expression values. Moreover, evaluation of sample quality at various steps in the data acquisition process is an important component of a multicenter study to ensure all participating centers adhere to established protocols.

SUBMITTER: Archer KJ

PROVIDER: S-EPMC3738008 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Quality assessment of microarray data in a multicenter study.

Archer Kellie J KJ Mas Valeria R VR O'Brien Thomas R TR Pfeiffer Ruth R Lum Nicole L NL Fisher Robert A RA

Diagnostic molecular pathology : the American journal of surgical pathology, part B 20090301 1

Issues implicit in a multicenter microarray study are protocol standardization and monitoring center adherence to established protocols. This study explored the effects of submitting center and sample preservation method on the quality of isolated RNA. In addition, the effects of sample preservation method and laboratory on microarray quality were also examined. Herein we evaluated the contribution of specific technical factors [center, laboratory, and preservation method (frozen/RNAlater)] on q ...[more]

PMID: 19214110

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Project description:Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n=223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals (42 healthy non-smokers, 49 healthy smokers, 11 symptomatic smokers, 22 smokers with lone emphysema with normal spirometry, and 20 smokers with COPD) were processed and hybridized to Affymetrix HG-U133 2.0 Plus microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) ≥7.0 using Agilent 2100 Bioanalyzer software; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets ≤3.0; and (3) the multi-chip normalization scaling factor ≤10.0 Of the 223 samples, 213 (95.5%) passed the QC criteria. In a data set of 34 arrays (10 samples failing QC criteria, 24 randomly chosen samples passing QC criteria), correlation coefficients for pairwise comparisons of expression levels for 100 housekeeping genes in which at least one array failed the QC criteria were significantly lower (average Pearson r = 0.90 ± 0.04) and more broadly dispersed than correlation coefficients for pairwise comparisons between any two arrays that passed the QC criteria (average Pearson r = 0.97 ± 0.01). By using the QC cutoff criteria, the inter-array variability, as assessed by the coefficient of variation in the expression levels for 100 housekeeping genes, was reduced from 35.7% to 21.7%. Based on the aberrant housekeeping gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation.

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Quality assessment of microarray data in a multicenter study.

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Quality assessment of microarray data in a multicenter study.

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