Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana.
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.
Wirta
Valtteri
V
Department of Molecular Biotechnology, KTH-Royal Institute of Technology, AlbaNova University Center, SE-106 91, Stockholm, Sweden. valtteri@biotech.kth.se
Holmberg
Anders
A
Lukacs
Morten
M
Nilsson
Peter
P
Hilson
Pierre
P
Uhlén
Mathias
M
Bhalerao
Rishikesh P
RP
Lundeberg
Joakim
J
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry*; D018744:DNA, Plant; D030541:Databases, Genetic; D015536:Down-Regulation; D019143:Evolution, Molecular; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D018745:Genome, Plant; D007091:Image Processing, Computer-Assisted; D007210:Indoleacetic Acids / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant; D019809:Streptavidin / Q000737:chemistry*; D015854:Up-Regulation
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D007210:Indoleacetic Acids; D012333:RNA, Messenger; D018749:RNA, Plant; D001710:Biotin; C030737:indoleacetic acid; D019809:Streptavidin