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.
*Virginia Commonwealth University, Richmond, VA
†Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda
‡Laboratory of Molecular Technology, National Cancer Institute, Frederick, MD
Supported by the National Institute of Diabetes and Digestive and Kidney Diseases 1RO1-DK069859 as well as by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Division of Cancer Epidemiology and Genetics.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
Reprints: Kellie J. Archer, PhD, Virginia Commonwealth University, 730 East Broad Street, Theater Row, 3-022, Richmond, VA 23298-0032 (e-mail: email@example.com).
Supplementary data available at http://www.molecularpathology.com.