Abstract
An improved cDNA-AFLP method for genome-wide expression analysis has been developed. We demonstrate that this method is an efficient tool for quantitative transcript profiling and a valid alternative to microarrays. Unique transcript tags, generated from reverse-transcribed messenger RNA by restriction enzymes, were screened through a series of selective PCR amplifications. Based on in silico analysis, an enzyme combination was chosen that ensures that at least 60% of all the mRNAs were represented by an informative sequence tag. The sensitivity and specificity of the method allows one to detect poorly expressed genes and distinguish between homologous sequences. Accurate gene expression profiles were determined by quantitative analysis of band intensities, and subtle differences in transcriptional activity were revealed. A detailed screen for cell cycle-modulated genes in tobacco demonstrates the usefulness of the technology for genome-wide expression analysis.
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Acknowledgements
We thank C. Tréhin for help with the BY2 synchronization experiments, M. Vuylsteke for advice on statistical evaluation of the data and valuable comments, and M. De Cock for help with the manuscript. K.V. is indebted to the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie for a predoctoral fellowship
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Communicated by R. G. Herrmann
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Breyne, P., Dreesen, R., Cannoot, B. et al. Quantitative cDNA-AFLP analysis for genome-wide expression studies. Mol Gen Genomics 269, 173–179 (2003). https://doi.org/10.1007/s00438-003-0830-6
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DOI: https://doi.org/10.1007/s00438-003-0830-6