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Sample GSM629079 Query DataSets for GSM629079
Status Public on Dec 31, 2019
Title mads-box mutant seeds, T3h of 5μM ABA, biological rep3
Sample type RNA
 
Source name Water-stratified Arabidopsis seeds, mads-box mutant
Organism Arabidopsis thaliana
Characteristics ecotype: Columbia-0 (Col-0)
genotype: mads-box mutant
tissue: seeds
treatment: water stratification, then abscisic acid (ABA) treatment
Treatment protocol Water-stratified seeds were transferred to light (22ºC, percival) in the presence of 5μM ABA for 3 hours.
Growth protocol Arabidopsis thaliana ecotype Columbia-0 (Col-0) and mads-box mutant seeds were stratified in water (4ºC, dark) for 3 days.
Extracted molecule total RNA
Extraction protocol RNA was isolated after Oñate-Sánchez and Vicente-Carbajosa (2008). Briefly, 30 - 50 mg ground seed powder was vortexed in 550 μl extraction buffer [0.4 M lithium chloride, 0.2 M Tris (hydroxymethyl)-methylamine (Tris base, pH = 8), 25 mM EDTA and 1% (w/v) sodium dodecyl sulphate] and 550 μl chloroform, and centrifuged (13,000 rpm) at room temperature for 3 min. Equal amounts of carbolic acid (phenol) and 1/6 of total volume of chloroform were added to the aqueous layer. Eight M LiCl was used to precipitate nucleic acids on ice for 3h. After centrifugation at 4 °C for 30 min (13,000 rpm) the pellet was resuspended in 470 μl diethylpyrocarbonate (DEPC)-treated water with 7 μl sodium acetate (3M, pH = 5.2) and 250 μl of 100 % ethanol to precipitate carbohydrates. To precipitate and further separate RNA from carbohydrates and proteins, the supernatant was transferred to a new tube and 43 μl sodium acetate (3 M, pH = 5.2) were added. After mixing, 750 μl of 100 % ethanol were added and kept at -20 °C for at least 1 h. The pellet was washed with 500 μl of 70 % ethanol and resuspended in 30 μl DEPC water. All the samples were purified using the RNeasy Mini Kit (Qiagen, Crawley, UK). RNA quantity and integrity were tested using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) together with an Agilent RNA 6000 Nano Labchip kit (Copois et al., 2007), generating an electropherogram and a gel-like image.
Label biotin
Label protocol cDNA was synthesized from 4 µg of total RNA using One-cycle target labeling and control reagents (Affymetrix, Santa Clara, CA) to produce biotin-labeled cRNA. The cRNA preparation (15 µg) were fragmented at 94ºC for 35 min into 35-200 bases in length.
 
Hybridization protocol If the quality control was correct, then 10 µg of fragmented cRNA were hybridized to the Arabidopsis ATH1 Genome array (Affymetrix, Santa Clara, CA), containing 22500 transcript variants from 24000 well-characterized Arabidopsis thaliana genes. Each sample was added to a hybridization solution containing 100mM 2-(N-morpholino) ethanesulfonic acid, 1 M Na+, and 20mM of EDTA in the presence of 0.01% of Tween-20 to a final cRNA concentration of 0.05 µg/ml. Hybridization was performed for 16 h at 45ºC. Each microarray was washed and stained with streptavidin-phycoerythrin in a Fluidics station 450 (Affymetrix). Three biological replicates for each condition were independently hybridized.
Scan protocol Each microarray was scanned at 2.5 µm resolution in a GeneChip Scanner 3000 7G System (Affymetrix).
Description Gene expression data from water-stratified mads-box mutant seeds, subsequently treated with 5μM ABA, before seed germination.
Data processing Analysis was performed using the affylmaGUI R package (Wettenhall et al, Bioinformatics (2006), 22, 897-899). The Robust Muti-array Analysis (RMA) algorithm was used for background correction, normalization and expression levels summarization (Irizarry et al. (2003) Biostatistics, 4, 249-264). Next, differential expression analysis was performed with the Bayes t-statistics from the linear models for Microarray data (limma), included in the affylmGUI package. P-values were corrected for multiple-testing using the Benjamini-Hochberg’s method (False Discovery Rate) (Benjamini, Y and Hochberg, Y (1995) J.Roy.Stat.Soc, 57, 289-300, Reiner et al. (2003) Bioinformatics, 19, 368-375). Genes were considered to be differentially expressed if the corrected P values were <0.05. In addition, only genes with a signal log2 ratio ≥1 for induction or ≤-1 for repression were considered for further analysis.
 
Submission date Nov 23, 2010
Last update date Dec 31, 2019
Contact name Oscar Lorenzo
E-mail(s) oslo@usal.es
Phone 34 923 294500 5117
Organization name Universidad de Salamanca
Department Dpt. Fisiologia Vegetal, Centro Hispano Luso de Investigaciones Agrarias (CIALE)
Lab lab. 7
Street address C/ Río Duero, 12 Campus de Villamayor. Parque Científico
City Salamanca
ZIP/Postal code 37185
Country Spain
 
Platform ID GPL198
Series (1)
GSE25451 Expression data from stratified mads-box (agl67) mutant and wild-type seeds, ABA-treated

Data table header descriptions
ID_REF
VALUE RMA signal intensity

Data table
ID_REF VALUE
244901_at 5.948708
244902_at 5.329663
244903_at 6.716435
244904_at 5.541105
244905_at 3.222026
244906_at 7.311709
244907_at 3.47373
244908_at 3.443836
244909_at 2.978274
244910_s_at 3.005004
244911_at 2.540763
244912_at 6.270431
244913_at 3.383174
244914_at 3.104112
244915_s_at 3.467123
244916_at 3.159619
244917_at 3.201504
244918_at 3.310373
244919_at 4.067503
244920_s_at 5.581205

Total number of rows: 22810

Table truncated, full table size 424 Kbytes.




Supplementary file Size Download File type/resource
GSM629079.CEL.gz 2.0 Mb (ftp)(http) CEL
Processed data included within Sample table

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