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Sample GSM205159 Query DataSets for GSM205159
Status Public on Aug 17, 2007
Title protoplast_KIN10_rep1
Sample type RNA
 
Source name isolated leaf mesophyll cells (protoplasts) transfected with KIN10 expressing plasmid DNA
Organism Arabidopsis thaliana
Characteristics Arabidopsis thaliana, Columbia-O ecotype, 28 days old plants
Treatment protocol For protoplast isolation, leaves were harvested 2h after the onset of the light period. Protoplasts (about 3 million) were freshly isolated from the fifth and sixth leaves of 36 randomly picked plants and pooled for two large protoplast-transfection experiments (scaled up 50-fold) as described (Kovtun et al., 2000, PNAS 97, 2940; Hwang and Sheen, 2001, Nature 413, 383; Sheen, 2001, Plant Physiol. 127, 1466). Cells (1.5 x 10-6) were transfected with 1mg of KIN10-expressing plasmid DNA and incubated in 10 ml mannitol buffer in a 150 x 15 mm Petri dish for 6h. Cells were kept at 23ºC under light (80 mmol/m2s) throughout the treatment period.
Growth protocol Plants were grown in soil (Metro Mix-360) under 13h light (80-100 mmol/m2s) /11 h dark conditions at 23ºC and 65% relative humidity.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol® (Invitrogen) following the manufacturer's protocol. The RNA was further purified using a Qiagen RNEasy Mini Kit.
Label Biotin-CTP and biotin-UTP
Label protocol Eight micrograms of purified total RNA was labeled for hybridization using an ENZO BioArray HighYield RNA Transcript Labeling Kit (T7) following standard Affymetrix protocols (Affymetrix GeneChip Technical Analysis Manual)
 
Hybridization protocol For each experimental sample, RNA quality was assessed by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Concentrations may also be determined using a NanoDrop 1000 spectrophotometer. Under standard conditions processing of RNAs for GeneChip Analysis was in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision four, as subsequently detailed. Synthesis of cDNA first and second strand is performed using the GeneChip Expression 3’-Amplification Reagents One-Cycle cDNA Synthesis Kit (P/N 900431). Cleanup of the double stranded product is carried according to standard Affymetrix protocols using the Affymetrix GeneChip Cleanup Module (Affymetrix Catalog # 900371).
In vitro transcription (IVT) is performed using the GeneChip Expression Amplification Reagents kit- 30 reactions (P/N 900449) and is carried out according to the standard Affymetrix protocols and quantification of the IVT samples is carried out on a Bio-Tek UV Plate Reader.
Hybridization is carried out according the Affymetrix GeneChip® Manual. Twenty micrograms of IVT material is the nominal amount used on the GeneChip® arrays. Affymetrix hybridization ovens are used to incubate the arrays at a constant temperature of 45°C overnight.
Scan protocol Preparation of microarrays for scanning is carried out with Affymetrix appropriate wash protocols matched to the specific chip type on a Model 450 Fluidics station. Affymetrix GeneChip Operating Software (GCOS) operating system controls the Fluidics station process.
Scanning is carried out on a GeneChip® Scanner 3000 7G scanner with autoloader. The Affymetrix GCOS v1.3 operating system controls the Model 3000 7G scanner and data acquisition functions. GCOS maintains the mediated first level data analysis and desktop data management for the entire GeneChip System. Chip library files specific to each array and necessary for scan interpretation are stored on the computer workstation controlling the scanner and are updated regularly as necessary when updates are made available from Affymetrix. Collected research data is stored on the hard drive of the instrument computer, transferred to a mirrored storage disk and to a raid 5 server operating on the Partners Healthcare network and backed up nightly via the Partners Healthcare Systems IT backup utility. All systems are CFR21-11 and HIPPA compliant.
Description Hybridization and scanning were performed at the Brigham and Women’s Hospital Microarray Facility (Boston, MA)
Data processing Transcript expression values from scanned arrays were imported and examined for quality control using Affymetrix GeneChip® Operating Software (GCOS v. 1.0). Values were subjected to global scaling using a target value of 1500, as suggested by Affymetrix.
 
Submission date Jun 22, 2007
Last update date Aug 28, 2018
Contact name Elena Baena-Gonzalez
E-mail(s) baena@molbio.mgh.harvard.edu
Organization name Massachusetts General Hospital and Harvard Medical School
Department Molecular Biology (MGH) and Department of Genetics (HMS)
Lab Jen Sheen
Street address Simches Research Building, 185 Cambridge Street, CPZN7250
City Boston
State/province MA
ZIP/Postal code 02114-2790
Country USA
 
Platform ID GPL198
Series (1)
GSE8257 Identification of KIN10-target genes in Arabidopsis mesophyll cells
Relations
Reanalyzed by GSE119083

Data table header descriptions
ID_REF
VALUE treatment (KIN10 expression) values after processing and scaling (1500) in GCOS
ABS_CALL detection call assigned by GCOS
DETECTION P-VALUE p-value associated with the detection call

Data table
ID_REF VALUE ABS_CALL DETECTION P-VALUE
244901_at 2424.5 P 0.000244
244902_at 2739.6 P 0.000244
244903_at 2432.4 P 0.000244
244904_at 612.1 P 0.001953
244905_at 117.2 A 0.665527
244906_at 1184 A 0.149658
244907_at 50.9 A 0.780518
244908_at 14.4 A 0.994141
244909_at 306.4 A 0.366211
244910_s_at 293.2 A 0.067627
244911_at 41.2 A 0.665527
244912_at 1899 P 0.01416
244913_at 136.4 A 0.665527
244914_at 7.7 A 0.633789
244915_s_at 15.1 A 0.888428
244916_at 79.1 A 0.567627
244917_at 14.3 A 0.953857
244918_at 176.9 A 0.466064
244919_at 30.8 A 0.601074
244920_s_at 1104.6 P 0.000732

Total number of rows: 22810

Table truncated, full table size 599 Kbytes.




Supplementary file Size Download File type/resource
GSM205159.CEL.gz 3.6 Mb (ftp)(http) CEL
Raw data included within Sample table
Processed data included within Sample table
Raw data provided as supplementary file

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