Bielefeld University,Institute for Genome Research and Systems Biology,Center for Biotechnology,D-33594 Bielefeld,GermanyMini-biographyHelge Küster heads the Junior Group "Genomics of Legume Plants" at the Institute for Genome Research, Center for Biotechnology, Bielefeld University. He obtained his PhD in Plant Genetics from Bielefeld University in 1995, at that time investigating genes expressed in Vicia faba root nodules. After switching his research interests towards the development and application of transcriptomics tools to study arbuscular mycorrhizal interactions, he received his Habilitation at the Faculty of Biology, Bielefeld University in 2004. Currently, H. Küster serves as "Head of the Scientific Committee" of the European Union Integrated Project
"Grain Legumes" , with a focus on integrating transcriptomics activities in different legume projects. His main involvement in GL-TTP relates to his involvement and interest in the construction of expression profiling tools that serve the needs of the legume communit
y.
Identification of candidate genes by transcriptomicsHelge Küster
Institute for Genome Research and Systems Biology, Center for Biotechnology (CeBiTec),
Bielefeld University, D-33594 Bielefeld, Germany
helge.kuester@genetik.uni-bielefeld.deThe initial regulation of gene expression is mediated by a cell- or condition-specific activation of promoters. At a single-gene level, the measurement of transcription was a key experiment ever since messenger RNAs were discovered in the late 1950s. In the last decade, high-throughput technologies were developed that allow the simultaneous measurement of gene expression not at the level of individual genes, but at the level of complete genomes. Such global profiling experiments not only provide genome-wide snapshots of gene expression, but also allow to select genes characterised by a specific expression pattern under a condition of interest, allowing the hypothesis that such a specific gene activation indicates a specific function. Since gene transcription is only the first step in the activation of genes, the simple conclusion that differential expression correlates with biological functions is not at all correct. Consequently, genes identified to be differentially transcribed are only candidates for genes with a possible relevance for conditions of interest, and subsequent functional studies using TILLING mutants or the generation of RNAi knock-down lines are important follow-ups.
This lecture will present the basics of major technologies currently available to call differentially expressed genes. Based on high-throughput cDNA-sequencing, expressed sequence tags (ESTs) can easily be generated for conditions of interest. Subsequently, these ESTs are usually clustered to derive minimal sets of tentative consensus sequences (TCs) that represent virtual genes. Based on the EST distribution, in silico analyses ("electronic Northerns") can be performed that provide collections of genes with a possible differential expression. In particular for species where a whole genome sequence is not available, TCs are not only good for gene identification, but also constitute a valuable resource for the construction of microarrays or chips. These experimental expression profiling tools allow the simultaneous measurement of activity for thousands of genes in a single experiment. In addition to microarrays that rely on nucleic acid hybridization, high-throughput real-time RT-PCR has recently emerged as a popular alternative, allowing an ultra-sensitive measurement of transcription by using gene-specific primers in a polymerase chain reaction (PCR)-based assay. The principles of these technologies and the different steps of evaluation of the basic data will be presented during the lecture.
In recent years, a range of public and project-specific databases emerged that allow to mine in silico and experimental expression data. Selected examples will be presented to demonstrate the power of integrated transcriptome databases for identification of candidate genes.
