Dye-bias correction in dual-labeled cDNA microarray gene expression measurements - Genomics and Risk Assessment: Mini-Monograph

Environmental Health Perspectives, March 15, 2004 by Barry A. Rosenzweig, P. Scott Pine, Olen E. Domon, Suzanne M. Morris, James J. Chen, Frank D. Sistare

A significant limitation to the analytical accuracy and precision of dual-labeled spotted cDNA microarrays is the signal error due to dye bias. Transcript-dependent dye bias may be due to gene-specific differences of incorporation of two distinctly different chemical dyes and the resultant differential hybridization efficiencies of these two chemically different targets for the same probe. Several approaches were used to assess and minimize the effects of dye bias on fluorescent hybridization signals and maximize the experimental design efficiency of a cell culture experiment. Dye bias was measured at the individual transcript level within each batch of simultaneously processed arrays by replicate dual-labeled split-control sample hybridizations and accounted for a significant component of fluorescent signal differences. This transcript-dependent dye bias alone could introduce unacceptably high numbers of both false-positive and false-negative signals. We found that within a given set of concurrently processed hybridizations, the bias is remarkably consistent and therefore measurable and correctable. The additional microarrays and reagents required for paired technical replicate dye-swap corrections commonly performed to control for dye bias could be costly to end users. Incorporating split-control microarrays within a set of concurrently processed hybridizations to specifically measure dye bias can eliminate the need for technical dye swap replicates and reduce microarray and reagent costs while maintaining experimental accuracy and technical precision. These data support a practical and more efficient experimental design to measure and mathematically correct for dye bias. Key words: cDNA, dye bias, dye swap, genomics, microarray. Environ Health Perspect 112:480-487 (2004). doi: 10.1289/txg.6694 available via http://dx.doi.org/[Online 15 January 2004]

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A frequent goal of genome-scale gene expression experiments is to identify significant alterations in transcript levels resulting from the exposure of a living system to a test agent at a given dose and time. For animal experimentation, biological replication is achieved through strategically paired dosings of individual animals. A study design involving, for example, a control group and three different test agent dose groups, each at two time points, can achieve three clear biological replicate measurements of each transcript on an array at each dose and time point using 24 animals. For a similar gene expression microarray experiment using cells in culture rather than animals, the designation of what constitutes a biological replicate is less clear. Does a biological replicate require a complete second experiment with cells expanded at some separate time, or could a replicate be considered a parallel set of additional culture-flask incubations arbitrarily assigned to a control or treatment group and treated and processed that same day? The volume of cultured cells needed to generate sufficient quantities of RNA for microarray gene expression analyses practically limits implementation of such an experiment to the pooling of multiple large flasks of cultured cells from each of the studied dose and time points. Eight samples of RNA would be collected for microarray analyses from such an experiment. To achieve the same minimum of three biological replicate measurements and therefore a similar level of confidence in biological accuracy, generally and practically speaking, this entire cell culture dosing experiment would be repeated on two additional separate occasions.

Technical replicate measurements of each of the individual biological replicates are generally incorporated at the discretion of the experimenter and may depend on factors including the amount of sample available, the budget of the experimenter, and whether the design of the specific microarray platform incorporates replicate probes. Replicate probes designed into a microarray platform to measure abundance of the same target gene transcript in a sample provide one approach to enhance confidence in the technical accuracy of relative transcript-level measurements. Repeat hybridizations of the same sample set using additional microarrays represent an additional level of technical replication to enhance confidence in the accuracy of each measurement of gene expression change. The precision and accuracy of replicated biological measurements is optimized when the technical replicate measurements are accurate and no confounding systematic error is introduced during sample processing.

Dual-labeled microarray hybridization protocols can introduce a systematic dye-bias error that could confound identification of true biological effects distinct from technical artifact (Goryachev et al. 2001; Ideker et al. 2000; Kerr et al. 2001; Tseng et al. 2001; Wang et al. 2001). Differences observed between red and green channel fluorescence intensities for a given transcript may be due to either a true biological difference resulting from the exposure of test agent to the cells or to a systematic bias resulting from individual transcript-dependent differences in efficiencies of dye incorporation and sample hybridizations.