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1 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
2 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA; Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
* To whom correspondence should be addressed. E-mail: mliang{at}mcw.edu.
Dye switching and biological replication substantially increase the cost and the complexity of experimental design and data analysis in cDNA microarray studies carried out using the two-color hybridization method. The objective of the present analysis was to quantitatively assess the importance of these procedures in order to provide a quantitative basis for decision making in the design of microarray experiments. Taking advantage of the unique characteristics of a previously published dataset, the impact of dye switching and/or biological replication was calculated using several indices of the reliability of microarray results. The results indicated that adding a second microarray with reverse labeling to examine the same pair of samples (i.e., dye switching) substantially increased the correlation coefficient between observed and predicted ln(ratio)s from 0.38±0.06 to 0.62±0.04 (N=12), and the outlier concordance from 21±3% to 43±4%. Dye switching also increased the correlation coefficient with the entire set of microarrays from 0.60±0.04 to 0.79±0.04 and the outlier concordance from 31±6% to 58±5%, and tended to improve the correlation with Northern blot analyses on 20 genes. Adding a replicate microarray without dye switching but examining a different pair of samples (i.e., biological replication) also improved the performance of these consistency indices, but often to a lesser degree. Simultaneous inclusion of dye switching and biological replication in the second microarray substantially improved the consistency with the entire set of microarrays, but had minimal effect on the consistency with predicted results. Analysis of another dataset generated using a different cDNA labeling method also supported a significant impact of dye switching on microarray results. In conclusion, both dye switching and biological replication substantially increased the reliability of microarray results, with dye switching likely having even greater benefits. Recommendations regarding the use of these procedures were proposed.
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