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Sample GSM1346523 Query DataSets for GSM1346523
Status Public on Mar 13, 2014
Title Clone#3_IgG_rep5
Sample type RNA
Source name ERK1-GFP human keratinocytes_clone#3_IgG
Organism Homo sapiens
Characteristics cell type: hTERT-immortalized normal human keratinocytes
tranfected with: ERK1-green fluorescent protein chimera
clone id: clone#3
treated with: nonspecific mouse IgG (control)
Treatment protocol Treatments were performed in KSF supplemented with bovine pituitary extract (no epidermal growth factor). Conditions included 10ng/mL of TGFα used for ligand-activation; 10ug/mL of monoclonal antibody EGF antagonist mAB225 was used to shut down autocrine-activated ERK1; and nonspecific mouse IgG was used as a control for the autocrine-activated ERK1. Cells were harvested for RNA extraction after 24hrs. Three replicates were collected.
Growth protocol hTERT-immortalized normal human keratinocytes (provided by Dr. Jerry Shay, The University of Texas Southwestern Medical Center); Maintained in Keratinocyte serum-free medium (KSF) supplemented with bovine pituitary extract and epidermal growth factor. Stable RNA transfection of ERK1-green fluorescent protein chimera was achieved using Phoenix retroviral packing cell system and puromycin selection. Stable subclones were isolated with distinct ERK activation/oscillation patterns: Clone #1 exhibits transient ERK activation with ligand activation but does not oscillate; Clone #2 exhibits persistent ERK oscillations that are dependent on ligand activation; and Clong #3 exhibits spontaneous ERK oscillations in the absence of ligand activation.
Extracted molecule total RNA
Extraction protocol Total RNA was collected using the RNeasy Kit (Qiagen, Valencia, CA).
Label biotin
Label protocol Complementary DNA was synthesized from 1 µg of total RNA in the presence of an oligo-dT primer containing a T7 RNA polymerase promoter, and an in vitro transcription reaction was performed in the presence of a mixture of biotin-labeled ribonucleotides to produce biotinylated cRNA from the cDNA template, according to manufacturer’s protocols (Affymetrix One-Cycle Target Labeling Kit).
Hybridization protocol Biotin-labeled cRNA (12 µg) was fragmented to a size range between 50-200 bases for array hybridization. After hybridization, the arrays were washed and stained with streptavidin-phycoerythrin.
Scan protocol The arrays were scanned at a resolution of 2.5 microns using an Affymetrix GeneChip Scanner 3000.
Description TW45
Clone #3 with autocrine activation
Data processing Raw intensity data were quantile normalized using Genespring software by Robust Multi-Array Analysis (RMA) summarization and probes were subject to quality control to measure the efficiency of transcription, integrity of hybridization, and consistency of qualitative calls.
Submission date Mar 12, 2014
Last update date Mar 13, 2014
Contact name Katrina M Waters
Organization name Pacific Northwest National Laboratory
Department Biological Sciences Division
Street address 902 Battelle Blvd; MSIN J4-18
City Richland
State/province WA
ZIP/Postal code 99352
Country USA
Platform ID GPL571
Series (1)
GSE55823 ERK Oscillation-Dependent Gene Expression Patterns and Deregulation By The Stress-Response

Data table header descriptions
VALUE Normalized Log2 signal intensity

Data table
AFFX-BioB-5_at 8.193467
AFFX-BioB-M_at 8.382479
AFFX-BioB-3_at 8.051421
AFFX-BioC-5_at 9.433744
AFFX-BioC-3_at 9.714991
AFFX-BioDn-5_at 10.744842
AFFX-BioDn-3_at 11.96629
AFFX-CreX-5_at 13.076174
AFFX-CreX-3_at 13.422686
AFFX-DapX-5_at 6.5789657
AFFX-DapX-M_at 8.347594
AFFX-DapX-3_at 8.864682
AFFX-LysX-5_at 4.0583153
AFFX-LysX-M_at 4.6685185
AFFX-LysX-3_at 5.5034113
AFFX-PheX-5_at 4.792664
AFFX-PheX-M_at 5.1835427
AFFX-PheX-3_at 6.5731196
AFFX-ThrX-5_at 5.8377976
AFFX-ThrX-M_at 5.274261

Total number of rows: 22277

Table truncated, full table size 443 Kbytes.

Supplementary file Size Download File type/resource
GSM1346523_TW45.CEL.gz 2.0 Mb (ftp)(http) CEL
Processed data included within Sample table

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