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Sample GSM344338 Query DataSets for GSM344338
Status Public on Nov 25, 2008
Title rat embryonic fibroblast cells sample3
Sample type RNA
 
Source name rat embryonic fibroblast cells
Organism Rattus norvegicus
Characteristics strain: DA
rat embryonic fibroblast cells
14.5 rat embryos
Extracted molecule total RNA
Extraction protocol Total RNA isolation from tissue: Cell culture is treated with 500μl of TriZol (Invitrogen,Carlsbad, CA) in a 1.5ml eppendorf and centrifuged for 20 min (4oC) at 12,000 x g. The supernatant is transferred to a fresh eppendorf and precipitated by the addition of 100ul chloroform and vortexed vigorously for 15 seconds. The sample is incubated for 3 minutes at room temperature and centrifuged for 15 minutes (4oC) at 10,000 x g. The aqueous phase is transferred to a fresh eppendorf and precipitated by the addition of 250μl isopropanol. The precipitate is incubated for 10 minutes at room temperature and centrifuged for 20 minutes (4oC) at 10,000 x g. The sample is washed once with 70% ethanol and allowed the pellet to air dry prior to resuspension in 100μl of DEPC treated water. The RNA is purified by Rneasy Clean up column (Qiagen, Valencia, CA).
Label biotin
Label protocol Prior to hybridization, 5.5μg of ssDNA is fragmented by incubation for 1hr at 37oC, follows by 2 min at 93oC. The 45μl mixture is added with 12μl of 5X TdT buffer, 2μl of TdT, 1μl of 5mM DNA labeling reagent, and is incubated at 37oC for 1hr, and follows by 70oC for 10 min.
 
Hybridization protocol Hybridization to the Affymetrix GeneChip Gene 1.0 ST Array is performed according to standard Affymetrix protocols. 5.5μg of fragmented and labeled DNA is added to 3.7μl of control oligonucleotide B2 (3nM), 11μl of 20x eukaryotic hybridization controls (bioB, bioC, bioD, and cre), 15.4μl of DMSO, 110μl of 2x hybridization buffer, and H2O to a final volume of 220μl. The hybridization cocktail is heated to 99oC for 5 min. An Affymetrix Gene 1.0 ST array is prepared by wetting for 10 min at 45oC with 1x hybridization buffer. The hybridization cocktail is transferred to a 45oC heat block for 5 min, and is clarified by centrifugation. The buffer solution is removed from the array and the clarified hybridization cocktail is added to the array cartridge.4 The array is hybridized for 16 hours in a 45oC rotisserie oven (60 rpm).
Scan protocol The hybridized array is washed, labeled with phycoerythrin conjugated streptavidin (Molecular Probes, Eugene, OR), scanned in the Affymetrix scanner signal detected and registered by Expression Console software
(Affymetrix).
Description rat embryonic fibroblast cell sample3
Data processing Extracted the expression data, performed normalization and transformation (Threshold all values to 5, Median Shift normalization to the 75 percentile, Baseline transformation using the median of all Samples) and Found Significant Differentially Expressed Genes. Unpaired t-test and Benjamini-Hochberg Correction were performed to produce table consisting of Probe Names, p-values, corrected p-values, Fold change (Absolute) and regulation. Fold change gives the absolute ratio of normalized intensities (no log scale) between the average intensities of the samples grouped.
 
Submission date Nov 20, 2008
Last update date Nov 24, 2008
Contact name Qilong Ying
E-mail(s) qying@usc.edu
Organization name USC
Department Broad Center for Regenerative Medicine and Stem Cell Research
Street address 1501 San Pablo St, ZNI 507
City Los Angeles
State/province CA
ZIP/Postal code 90033
Country USA
 
Platform ID GPL6247
Series (1)
GSE13681 Genome wide analysis of gene expression of rat ES cells, rat embryonic fibroblast cells and mouse ES cells

Data table header descriptions
ID_REF
VALUE rma-gene-default-Signal

Data table
ID_REF VALUE
10700001 12.042
10700002 7.41327
10700003 10.6581
10700004 5.93842
10700005 9.66613
10700006 2.36998
10700007 2.60771
10700008 2.231
10700009 9.49094
10700010 3.01917
10700011 5.1447
10700012 4.20729
10700013 11.1962
10700014 10.5845
10700015 9.75553
10700016 2.37576
10700017 6.65491
10700018 2.20062
10700019 2.43912
10700020 12.0409

Total number of rows: 29215

Table truncated, full table size 481 Kbytes.




Supplementary file Size Download File type/resource
GSM344338.CEL.gz 4.1 Mb (ftp)(http) CEL
GSM344338.chp.gz 228.2 Kb (ftp)(http) CHP
Processed data included within Sample table
Processed data provided as supplementary file

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