Abstract
Microarrays are a powerful tool for comparison and understanding of gene expression levels in healthy and diseased states. The method relies upon the assumption that signals from microarray features are a reflection of relative gene expression levels of the cell types under investigation. It has previously been reported that the classical fluorescent dyes used for microarray technology, Cy™3 and Cy™5, are not ideal due to the decreased stability and fluorescence intensity of the Cy™5 dye relative to the Cy™3, such that dye bias is an accepted phenomena necessitating dye swap experimental protocols and analysis of differential dye affects. The incentive to find new fluorophores is based on alleviating the problem of dye bias through synonymous performance between counterpart dyes. Alexa Fluor® 555 and Alexa Fluor® 647 are increasingly promoted as replacements for CyDye™ in microarray experiments. Performance relates to the molecular and steric similarities, which will vary for each new pair of dyes as well as the spectral integrity for the specific application required. Comparative analysis of the performance of these two competitive dye pairs in practical microarray applications is warranted towards this end. The findings of our study showed that both dye pairs were comparable but that conventional CyDye™ resulted in significantly higher signal intensities (P < 0.05) and signal minus background levels (P < 0.05) with no significant difference in background values (P > 0.05). This translated to greater levels of differential gene expression with CyDye™ than with the Alexa Fluor® counterparts. However, CyDye™ fluorophores and in particular Cy™5, were found to be less photostable over time and following repeated scans in microarray experiments. These results suggest that precautions against potential dye affects will continue to be necessary and that no one dye pair negates this need.
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References
Al-Mulla, F., Al-Tamimi, R., & Bitar, M. S. (2004). Comparison of two probe preparation methods using long oligonucleotide microarrays. BioTechniques, 37, 827–833.
Amersham Biosciences (2002). Microarray handbook. Buckinghamshire, UK: . Amersham Biosciences Corp.
Berlier, J. E., Rothe, A., Buller, G., Bradford, J., Gray, D. R., Filanoski, B. J., Telford, W. G., & Yue, S., et al. (2003). Quantitative comparison of long-wavelength Alexa Fluor® dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. Journal of Histochemistry and Cytochemistry, 51, 1699–1712.
Cox, W. G., & Singer, V. L. (2004). Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling. BioTechniques, 36, 114–122.
Cox, W. G., Beaudet, M. P., Agnew, J. Y., & Ruth, J. L. (2004). Possible sources of dye-related signal correlation bias in two-color DNA microarray analysis. Analytical Biochemistry, 331, 243–254.
Dobbin, K. K., Kawasaki, E. S., Petersen, D. W., & Simon, R. M. (2005). Characterizing dye bias in microarray experiments. Bioinformatics, 21, 2430–2437.
Forster, T., Costa, Y., Roy, D., Cooke, H. J., & Maratou, K. (2004). Triple-target microarray experiments: a novel experimental strategy. B.M.C. Genomics, 5, e13.
Gruber, H. J., Hahn, C. D., Kada, G., Riener, C. K., Harms, G. S., Ahrer, W., Dax, T. G., & Knaus, H. G. (2000). Anomalous fluorescence enhancement of Cy3 and Cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin. Bioconjugate Chemistry, 11, 696–704.
Lam, C. W., Cheung, K. K., Luk, N. M., Chan, S. W., Lo, K. K., & Tong, S. F. (2005). DNA-based diagnosis of xeroderma pigmentosum group C by Whole-genome scan using single-nucleotide polymorphism microarray. Journal of Investigative Dermatology, 124, 87–91.
Nguyen, D. V., Arpat, A. B., Wang, N., & Carroll, R. J. (2002). DNA microarray experiments: biological and technical aspects. Biometrics, 58, 701–717.
Panchuk-Voloshina, N., Haugland, R. P., Bishop-Stewart, J., Bhalgat, M. K., Millard, P. J., Mao, F., Leung, W. Y., & Haugland, R. P. (1999). Alexa Dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. Journal of Histochemistry and Cytochemistry, 47, 1179–1188.
Peeters, J. K., & Van der Spek, P. J. (2005). Growing applications and advancements in microarray technology and analysis tools. Cell Biochemistry and Biophysics, 43, 149–166.
Shang, S., Chen, G., Wu, Y., Du, L., & Zhao, Z. (2005). Rapid diagnosis of bacterial sepsis with PCR amplification and microarray hybridization in 16S rRNA gene. Pediatric Research, 58, 143–148.
Staal, Y. C., van Herwijnen M. H. M., van Schooten F. J., & van Delft J. H. M. (2005). Application of four dyes in gene expression analysis by microarrays. BMC Genomics, 6, 101–113.
Takahashi, M., Yang, X. J., McWhinney, S., Sano, N., Eng, C., Kagawa, S., Teh, B. T., & Kanayama, H. O. (2005). cDNA microarray analysis assists in diagnosis of malignant intrarenal pheochromocytoma originally masquerading as a renal cell carcinoma. Journal of Medical Genetics, 42, e48.
Tseng, G. C., Oh, M. K., Rohlin, L., Liao, J. C., & Wong, W. H. (2001). Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects. Nucleic Acids Research, 29, 2549–2557.
Wildsmith, S. E., Archer, G. E., Winkley, A. J., Lane, P. W., & Bugelski, P. J. (2001). Maximization of signal derived from cDNA microarrays. BioTechniques, 30, 202–208.
Yu, J., Othman, M. I., Farjo, R., Zareparsi, S., MacNee, S. P., Yoshida, S., & Swaroop, A. (2002). Evaluation and optimization of procedures for target labeling and hybridization of cDNA microarrays. Molecular Vision, 8, 130–137.
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We thank the Laboratory for Molecular Genetics at the Western Australian Institute for Medical Research for their donation of HEK cells and the Tumour Immunology Group at the School of Medicine and Pharmacology of The University of Western Australia who donated JU77 cells. We gratefully thank and acknowledge Lotterywest for their funding of the LSMAF and this study.
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Ballard, J.L., Peeva, V.K., deSilva, C.J.S. et al. Comparison of Alexa Fluor® and CyDye™ for practical DNA microarray use. Mol Biotechnol 36, 175–183 (2007). https://doi.org/10.1007/s12033-007-0006-4
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DOI: https://doi.org/10.1007/s12033-007-0006-4