Unknown,Transcriptomics,Genomics,Proteomics

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Expression profile in response to salt-treatment in wheat using 22k microarray

ABSTRACT: Analysis of transcripts in response to salt treatment. In order to design the 22k wheat oligo-DNA microarray, a total of 148,676 expressed sequence tags of common wheat were collected from the database of the Wheat Genomics Consortium of Japan. These were grouped into 34,064 contigs, which were then used to design an oligonucleotide DNA microarray. Following a multi-step selection of the sense strand, 21,939 60-mer oligo DNA probes were selected for attachment on the microarray slide. This 22k oligo DNA microarray was used to examine the transcriptional response of wheat to salt stress. More than 95% of the probes gave reproducible hybridization signals when targeted with RNAs extracted from salt-treated wheat shoots and roots. With the microarray, we identified 1,811 genes whose expressions changed more than two-fold in response to salt. These included genes known to mediate the response to salt as well as unknown genes, and they were classified into 12 major groups by hierarchical clustering. These gene expression patterns were also confirmed by real-time reverse transcription (RT)-PCR. Many of the genes with unknown function were clustered together with genes known to be involved in the response to salt stress. Thus, analysis of gene expression patterns combined with gene ontology should help identify the function of the unknown genes. Also, functional analysis of these wheat genes should provide new insight into the response to salt stress. Finally, these results indicate that the 22k oligo DNA microarray is a reliable method for monitoring global gene expression patterns in wheat. Microarray hybridization was performed by a competitive two-color method including color-swap experiments. Chinese Spring wheat was grown for two weeks and treated with 150mM NaCl for 0, 1, 6 and 24 hours. RNA samples were extracted from roots and shoots.Each experiment was design for comparison in control and treated sample.

ORGANISM(S): Triticum aestivum

SUBMITTER: Yasunari Ogihara

PROVIDER: E-GEOD-8060 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A microarray analysis of wheat grain hardness For both the Heron and Falcon NIL, whole wheat heads were harvested at 6, 15 and 25 days post anthesis (DPA) from glass house grown material and stored at -80 OC. For the other wheat cultivars analysed, heads at approximately 9-10 DPA were used. Eight to ten developing caryopses at the same age and morphological stage of development were selected from a head to represent a sample. Seed collected from a different head represented a replicate sample. Total RNA was isolated from the whole seed following the method of Higgins et al. (1976) with the following modification to remove starch. After the lithium chloride precipitation, the RNA pellet was resuspended in 250 ?l water. Then 3.5 ?l of 3 M sodium acetate (pH 5.3) and 125 ?l ethanol were added and the sample was centrifuged for 10 minutes at 4 OC. The supernatant was transferred to a fresh tube and the RNA was ethanol-precipitated by the addition of 21.5 ?l of 3 M sodium acetate (pH 5.3) and 375 ?l ethanol. The recovery of RNA varied from 0.06-0.5 ?g RNA per mg of tissue and this was dependent on the developmental stage of the seeds used for extraction of the RNA. For the labelling procedure, 50 ?g of total RNA was used for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), following the two-step labelling method of Schenk et al. (2000). The Cy3 control (hard wheat) and Cy5 sample (soft wheat) were swapped among the replicate experiments to minimise any bias in cDNA incorporation and photo-bleaching of the fluorescent dyes. The pre-hybridisation of the microarray slides, hybridisation of the target cDNA and subsequent washing of the slides to remove unbound target was performed as per the supplied protocol for the CMT-GAPSTM coated microscope slides (Corning USA). The slides were scanned with a GenePix 4000A microarray scanner (Axon Instruments, Union, CA, USA). The features were analysed using the GenePix Pro 4 software and unsatisfactorily segmented features were either manually adjusted or discarded to ensure the integrity of the data obtained.

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Expression profile in response to salt-treatment in wheat using 22k microarray

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