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Sample GSM111147 Query DataSets for GSM111147
Status Public on Mar 25, 2007
Title genotype RL4452 developing seed at 5 days after anthesis, biological rep2
Sample type RNA
 
Source name developing seed collected from field grown material 5 days after anthesis
Organism Triticum aestivum
Characteristics Genotype: RL4452, 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 μL 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°C 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°C. 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°C. 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°C overnight then centrifuged at 14,000 rpm at 4°C 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 Replicate 2
Data processing RMA-calculated, normalized to median signal intensity
 
Submission date May 25, 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)
GSE4929 wheat expression level polymorphism study parental 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 271.06995
AFFX-BioB-M_at 154.6325
AFFX-BioB-3_at 136.99948
AFFX-BioC-5_at 464.26932
AFFX-BioC-3_at 528.13
AFFX-BioDn-5_at 1079.217
AFFX-BioDn-3_at 2643.1658
AFFX-CreX-5_at 5884.885
AFFX-CreX-3_at 7624.8433
AFFX-DapX-5_at 415.6176
AFFX-DapX-M_at 850.8504
AFFX-DapX-3_at 1571.7745
AFFX-LysX-5_at 48.478527
AFFX-LysX-M_at 98.89031
AFFX-LysX-3_at 251.12897
AFFX-PheX-5_at 101.97398
AFFX-PheX-M_at 127.87683
AFFX-PheX-3_at 249.31873
AFFX-ThrX-5_at 160.70284
AFFX-ThrX-M_at 217.61069

Total number of rows: 61290

Table truncated, full table size 1726 Kbytes.




Supplementary file Size Download File type/resource
GSM111147.CEL.gz 7.9 Mb (ftp)(http) CEL
GSM111147.EXP.gz 381 b (ftp)(http) EXP
Raw data provided as supplementary file

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