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Series GSE2624 Query DataSets for GSE2624
Status Public on Jul 04, 2005
Title TNF time course study
Organism Homo sapiens
Experiment type Expression profiling by array
Summary TNF time course series of HelA tet off cells cultured in presence or absence of Dox

TNF is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the Nuclear Factor-kB (NF-kB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kB dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kB inhibitor to systematically identify NF-kB dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kB signaling was analyzed.

Methods:
Cell culture, treatment and transfection-The human cervical epithelioid carcinoma cell line HeLa expressing tTA (HeLa Tet-Off) pBI-EGFP- IkBa Mut was constructed earlier (23). HeLa Tet-Off were grown in medium containing 90 % Dulbecco’s Modified Eagle’s Medium (DMEM), 10 % heat-inactivated fetal bovine serum (FBS), 4mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mg/ml G418, 100 units /ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5 % CO2. For TNF stimulation, freshly isolated cells were split into two cultures, one group maintained in Dox, the other without for 7 d, a time at which IkBa Mut expression was maximal. Thereafter, recombinant human (rh) TNFa (25 ng/ml, final concentration) was added directly to the culture medium for indicated times prior to harvest.


Oligonucleotide probe based microarray-Hu95Av2 GeneChip (Affymetrix Inc, Santa Clara, CA) containing 12,626 sequenced human genes were hybridized according to the manufacturer’s recommendations and washed using both non-stringent (1 M NaCl, 25 °C) and stringent (1 M NaCl, 50 °C) conditions prior to staining with phycoerythrin streptavidin (10 mg/ml final). Arrays were scanned using a Gene Array Scanner (Hewlett Packard) and analyzed using the Gene Chip Analysis Suite 5 software (Affymetrix Inc). For each gene, 16-20 probe pairs are immobilized as ~25-mer oligonucleotides that hybridize throughout the mRNA; each probe pair is represented as a perfect match (PM) oligonucleotide and a mismatch (MM) oligonucleotide as hybridization control. The the Absolute Call [e.g., the gene is detected (“present”) or not (“absent”)] and the Signal Intensity (measure of mRNA abundance) is determined.

Four independent experiments were performed identically including control (0 h ), 1, 3 and 6 h TNF stimulation (25 ng/ml) in the presence or absence of Doxycyline (2 mg/ml) in growth medium.
These are indicated as 180, 181, 220 and 266 series, and are considered replicates of each other.
Keywords: time-course
 
 
Contributor(s) Brasier AR, Tian B
Citation(s) 15722553
Submission date May 04, 2005
Last update date Dec 13, 2018
Contact name Allan Brasier
E-mail arbrasie@utmb.edu
Phone 409-772-2824
Fax 409-772-8709
Organization name University Texas Medical Branch
Department Medicine
Street address 301 Univ. Blvd
City Galveston
State/province TX
ZIP/Postal code 77555-1060
Country USA
 
Platforms (1)
GPL8300 [HG_U95Av2] Affymetrix Human Genome U95 Version 2 Array
Samples (32)
GSM50270 180C0
GSM50297 180C1
GSM50298 180C3
Relations
BioProject PRJNA92135

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