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AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis

Abstract

Although DNA microarrays are currently the standard tool for genome-wide expression analysis, their application is limited to organisms for which the complete genome sequence or large collections of known transcript sequences are available. Here, we describe a protocol for cDNA-AFLP, an AFLP-based transcript profiling method that allows genome-wide expression analysis in any species without the need for prior sequence knowledge. In essence, the cDNA-AFLP method involves reverse transcription of mRNA into double-stranded cDNA, followed by restriction digestion, ligation of specific adapters and fractionation of this mixture of cDNA fragments into smaller subsets by selective PCR amplification. The resulting cDNA-AFLP fragments are separated on high-resolution gels, and visualization of cDNA-AFLP fingerprints is described using either a conventional autoradiography platform or an automated LI-COR system. Observed differences in band intensities between samples provide a good measure of the relative differences in the gene expression levels. Identification of differentially expressed genes can be accomplished by purifying cDNA-AFLP fragments from sequence gels and subsequent sequencing. This method has found widespread use as an attractive technology for gene discovery on the basis of fragment detection and for temporal quantitative gene expression analysis. The protocol can be completed in 3–4 d.

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Figure 1: Outline of the 'one-gene–one-tag' complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) procedure using the BstYI/MseI restriction enzyme combination (EC):
Figure 2: Schematic for adapter and primer design for the rare cutter, BstYI, and the two frequent cutters MseI and TaqI.
Figure 3: Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) fingerprint of two Catharanthus roseus transgenic genotypes.
Figure 4: Electropherogram with representative examples of complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) sequence and expression polymorphisms (SPs and EPs).

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Acknowledgements

We are grateful to H. Van den Daele and I. Vercauteren for their help in writing the manuscript, and to Alain Goossens for being so kind as to provide the gel image of C. roseus. The AFLP and cDNA-AFLP technology are covered by patents and patent applications owned by Keygene N.V. AFLP and AFLP-QuantarPro are registered trademarks of Keygene N.V. All other product names, brand names or company names are used for identification purposes only and may be (registered) trademarks of their respective owners.

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Correspondence to Marnik Vuylsteke.

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M.J.T. van Ejik and J.D. Peleman are full-time employees of Keygene N.V.

M. Vuylsteke declares not to have competing interests.

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Vuylsteke, M., Peleman, J. & van Eijk, M. AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis. Nat Protoc 2, 1399–1413 (2007). https://doi.org/10.1038/nprot.2007.174

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