Abstract
BackgroundTranscriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific.ResultsWe present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes.ConclusionsThe described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21 000 Arabidopsis transcripts.
Highlights
Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation
The design of the CATMA gene sequence tag (GST) probes is based on de novo gene prediction from the genome sequence [1,3,4], since only a relatively number of expressed sequence tags (ESTs) are available for Arabidopsis
The purification method is based on reversible biotin-streptavidin binding, utilises streptavidin-coated paramagnetic beads and can be automated on a robotic workstation dedicated for magnetic separation and equipped with a temperature control [6]
Summary
Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as crosshybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific. In a recent study the CATMA, Affymetrix and Agilent arrays were found to perform but with a minor advantage for the CATMA arrays in terms of dynamic range [5]
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