Abstract
Hybridization of labeled cDNA to microarrays is an intuitively simple and a vastly underestimated process. If it is not performed, optimized, and standardized with the same attention to detail as e.g., RNA amplification, information may be overlooked or even lost. Careful balancing of the amount of labeled cDNA added to each slide reduces dye-bias and slide to slide variation. Efficient mixing of the hybridization solution throughout the hybridization reaction increases signals several fold. The amount of near perfect target–probe hybrids may be reduced by efficient stringency washes of the hybridized microarray slides.
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Acknowledgments
Above all I would like to express my sincere gratitude to Dr. Martin Dufva for giving me the opportunity to share my hard earned experiences with the community. I wished for a collection of relevant protocols including tips and tricks when I started out my experiments, but I found none. I hope some may benefit from reading this.
A grant by the Danish Biotechnology Instrument Center (DABIC) project no. 2014-00-0003 financially supported the project. This is gratefully acknowledged.
Last but not least – my family, especially my wife, Lene. Without her ever present support and tolerance for my physical and mental absence, my completion of this project would not have been possible.
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Stangegaard, M. (2009). Gene Expression Analysis Using Agilent DNA Microarrays. In: Dufva, M. (eds) DNA Microarrays for Biomedical Research. Methods in Molecular Biology, vol 529. Humana Press. https://doi.org/10.1007/978-1-59745-538-1_9
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DOI: https://doi.org/10.1007/978-1-59745-538-1_9
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