Robust interlaboratory reproducibility of a gene expression signature measurement consistent with the needs of a new generation of diagnostic tools

Robert A Ach,Annuska M Glas,Anya Tsalenko,Fatima Cardoso,Laura J Van'T Veer,Bo Curry,Vladimir Lazar,Arno Floore,Laurakay Bruhn,Rob Pover,Hugues Ripoche,Mahasti D'Assignies

doi:10.1186/1471-2164-8-148

Abstract

BackgroundThe increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. While the reproducibility of microarray gene expression measurements has been the subject of several recent reports, there is still a need for systematic investigation into what factors most contribute to variability of measured expression levels observed among different laboratories and different experimenters.ResultsWe report the results of an interlaboratory comparison of gene expression array measurements on the same microarray platform, in which the RNA amplification and labeling, hybridization and wash, and slide scanning were each individually varied. Identical input RNA was used for all experiments. While some sources of variation have measurable influence on individual microarray signals, they showed very low influence on sample-to-reference ratios based on averaged triplicate measurements in the two-color experiments. RNA labeling was the largest contributor to interlaboratory variation.ConclusionDespite this variation, measurement of one particular breast cancer gene expression signature in three different laboratories was found to be highly robust, showing a high intralaboratory and interlaboratory reproducibility when using strictly controlled standard operating procedures.

Highlights

The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements
One factor that will influence the capability to fully realize the potential utility of these signatures for biomedical research, toxicogenomics, pharmaceutical development, and diagnostics is the reproducibility of the technology used to measure them
The Microarray Quality Control project (MAQC) compared gene expression measurements of two RNA samples using a number of microarray platforms, as well as alternative technologies, and demonstrated intraplatform consistency and interplatform concordance in terms of genes differentially expressed [20]

Summary

Introduction

The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. A second study measured gene expression in a set of four knockout human cell lines across ten laboratories using three different microarray platforms [18] They found that the particular laboratory which performed the analysis had a greater effect on the precision than did the choice of platform, and the results from the best-performing labs agreed fairly well. A related study found consistency among microarray platforms at different sites using 36 different RNAs from rats treated with three chemicals [21] Neither of these two recent studies examined whether the variation seen between laboratories was due to the labeling or hybridization steps, or both. While these papers give a general overview of the reproducibility of microarraybased gene expression profiling across a variety of platforms, they focused on the overall reproducibility of measurements made with arrays containing probes designed to measure the majority of known human transcripts, rather than on the reproducibility of gene expression signatures composed of relatively small numbers of genes analyzed on a smaller, targeted array

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Conclusion