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Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-3811-5439
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-2745-6289
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0002-4657-8532
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2005 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 5, 5- p.Article in journal (Refereed) Published
Abstract [en]

Background: Transcriptional 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.

Results: We 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.

Conclusions: The 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.

Place, publisher, year, edition, pages
2005. Vol. 5, 5- p.
Keyword [en]
Arrays, Genes, Image processing, Nucleic acids, Optimization, Probes, Purification
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-6165DOI: 10.1186/1472-6750-5-5ISI: 000227244000001Scopus ID: 2-s2.0-26844497995OAI: oai:DiVA.org:kth-6165DiVA: diva2:10797
Note
QC 20100831Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Mining the transcriptome - methods and applications
Open this publication in new window or tab >>Mining the transcriptome - methods and applications
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods.

The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal.

This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype.

To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 62 p.
Series
Theses in philosophy from the Royal Institute of Technology, ISSN 1650-8831
Keyword
transcriptome, gene expression profiling, EST, microarray, RNA amplification, stem cells, neurosphere
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4115 (URN)91-7178-436-5 (ISBN)
Public defence
2006-10-13, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
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Supervisors
Note
QC 20100927Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2010-09-27Bibliographically approved

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Wirta, ValtteriHolmberg, AndersUhlén, Mathias

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