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Sample GSM111230 Query DataSets for GSM111230
Status Public on Mar 25, 2007
Title genotype B33 developing seed at 5 days after anthesis, biological rep1
Sample type RNA
 
Source name developing seed collected from field grown material 5 days after anthesis
Organism Triticum aestivum
Characteristics Genotype: B33 from RL4452 x AC Domain mapping population
Age: developing seed 5 days after anthesis
Treatment protocol spikes were tagged when anthers were visible, enough developing seeds to fill a 2mL tube were isolated from each spike 5 days after tagging and flash frozen in liquid nitrogen
Growth protocol The 39 lines and parental lines were grown as 1.47 m rows in three replications in a 7x7 (49 entry) lattice design at Winnipeg, Manitoba in 2004. Each replicate had a unique randomization
Extracted molecule polyA RNA
Extraction protocol Total RNA was extracted from frozen seed using a modification of the protocol described at http://plantsciences.montana.edu/wheat-transformation/molecular.htm. The content of a 2 mL tube full of frozen seeds was ground to a fine powder in liquid nitrogen using a mortar and pestle. Ground seed was transferred to 50 mL Oak Ridge tubes and 7.5 mL of extraction buffer was added. [50 mL Extraction Buffer contained 35 mL nuclease-free water, 2.5 mL 1M Tris, pH 9.0. 5 mL 2M NaCl, 5 mL 10% Sarcosyl, 2 mL 0.5M EDTA, pH 8.0, 250 ?L 1M DTT]. After vortexing well, 3.75 mL phenol and 3.75 mL chloroform/isoamyl alcohol (49:2) were added and samples vortexed again. Samples were centrifuged 10 min at 13,000 rpm in a Sorvall model RC5C at 4 using an SS-34 rotor. A total of 7.5 mL of the upper aqueous phase was transferred to a clean Oak Ridge tube, 15 mL Trizol was added and the sample was vortexed. A volume of 3 mL of choloroform was added and the sample was vortexed again. Samples were centrifuged 10 min at 13,000 rpm in the Sorvall at 4. A volume of 15 mL of the upper aqueous phase was transferred to a fresh Oak Ridge tube and 10 mL of chloroform was added and the sample was vortexed. Samples were centrifuged again in the Sorvall at 13,000 rpm for 10 min at 4. A volume of 12 mL of the upper phase was transferred to a fresh Oak Ridge tube and the RNA precipitated by adding 1,200 uL of 3M sodium acetate and 24 mL of absolute ethanol. Samples were left at -80 overnight then centrifuged at 14,000 rpm at 4 in the Sorvall for 20-30 minutes to precipitate the RNA. The pellet was rinsed with 70% ethanol, lightly dried and re-suspended in 1 mL of nuclease-free water. RNA concentration was quantified using a spectrophotometer. All solutions and equipment used in this procedure were RNAse-free. Messenger RNA was enriched from total RNA using the Poly(A) Purist MAG kit (Ambion, http://www.ambion.com) according to the protocol provided by the manufacturer. Messenger RNA was checked for quality and quantified by micro-chromatography using an Agilent 2000 Bioanalyzer and the RNA 6000 Nano Assay Kit both purchased from Agilent Technologies (http://www.agilent.com). Quantification was carried out according to the instructions provided with the kit.
Label biotin
Label protocol Biotinylated cRNA were prepared according to the standard Affymetrix protocol (Expression Analysis Technical Manual, Affymetrix). mRNA was used for first and second strand cDNA synthesis using the One Cycle cDNA Synthesis Kit (Affymetrix, http://www.affymetrix.com) following the manufacturers instructions. The resulting cDNA was purified using the GeneChip Sample Cleanup Module (Affymetrix) according to instructions and then used to make purified biotin labelled cRNA using the GeneChip IVT labelling kit and the GeneChip Sample Cleanup Module (Affymetrix). The resulting labelled cRNA was quantified and the yield and size distribution of labelled fragments checked by electrophoresis using the Agilent Bioanalyzer. The labelled cRNA was fragmented using the GeneChip Sample Cleanup Module (Affymetrix) and stored at -20°C.
 
Hybridization protocol Following fragmentation, the labelled samples were mixed with 0.1 mg/ml sonicated herring sperm DNA in hybridization buffer (100mM 2-N-morpholino-ethane-sulphonic acid (MES), 1 M NaCL, 20mM EDTA, 0.01% Tween 20, 0.5 mg/ml BSA, 10% DMSO), denatured at 99°C for 5 min then added to GeneChip Wheat Genome Array (Affymetrix) according to manufacturer instructions and hybridized for 16 h at 45°C in a rotisserie oven at 60 rpm. Following hybridization the arrays were washed and stained in the Affymetrix fluidics station 450 according to standard protocol (Section 2.3.3 of the Expression Analysis Technical Manual (http://www.affymetrix.com/support/technical/manual/expression_manual.affx)
Scan protocol GeneChips were scanned using the Affymetrix scanner 3000.
Description Gene expression data from field grown developing seeds 5 days after anthesis
Data processing The raw data in the Affymetrix CEL files of all 78 arrays representing two biological replicates of each of 39 genotypes were subjected to quantile normalization using Robust Multi-Array Average (RMA) (Bolstad, B.M., Irizarry, R.A., Astrand, M., Speed, T.P. 2003. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185-193) followed by normalization to the median.
 
Submission date May 26, 2006
Last update date Sep 28, 2006
Contact name Mark Jordan
E-mail(s) mcjordan@agr.gc.ca
Organization name Agriculture and Agri-Food Canada
Department Cereal research Centre
Street address 195 Dafoe Rd.
City Winnipeg
State/province Manitoba
ZIP/Postal code R3T 2M9
Country Canada
 
Platform ID GPL3802
Series (1)
GSE4935 wheat expression level polymorphism study 39 genotypes 2 biological reps

Data table header descriptions
ID_REF
VALUE RMA-calculated, normalized to median signal intensity

Data table
ID_REF VALUE
AFFX-BioB-5_at 368.02124
AFFX-BioB-M_at 260.29654
AFFX-BioB-3_at 285.58224
AFFX-BioC-5_at 750.49164
AFFX-BioC-3_at 778.98395
AFFX-BioDn-5_at 1695.1813
AFFX-BioDn-3_at 4001.3796
AFFX-CreX-5_at 9914.443
AFFX-CreX-3_at 11463.681
AFFX-DapX-5_at 454.30972
AFFX-DapX-M_at 1185.5719
AFFX-DapX-3_at 2506.948
AFFX-LysX-5_at 114.25229
AFFX-LysX-M_at 138.6282
AFFX-LysX-3_at 384.72012
AFFX-PheX-5_at 145.00766
AFFX-PheX-M_at 211.1388
AFFX-PheX-3_at 338.7184
AFFX-ThrX-5_at 232.19432
AFFX-ThrX-M_at 380.49353

Total number of rows: 61290

Table truncated, full table size 1727 Kbytes.




Supplementary file Size Download File type/resource
GSM111230.CEL.gz 7.9 Mb (ftp)(http) CEL
GSM111230.EXP.gz 496 b (ftp)(http) EXP
Raw data provided as supplementary file

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