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Sample GSM142373 Query DataSets for GSM142373
Status Public on Oct 31, 2006
Title GATA5 (-/-) -RA vs +RA
Sample type RNA
 
Channel 1
Source name GATA5 (-/-) RW-4 ES cells
Organism Mus musculus
Characteristics GATA5 (-/-) RW-4 ES cells, untreated
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from 60-80% confluent ES cell cultures. Total RNA was isolated using the guanidinium/isothiocyanate/phenol/chloroform method as previously described (Schultz). Total RNA was DNase treated using “DNA free” kit (Ambion, Austin, TX) according to manufacturer’s specifications.
Label Cy5
Label protocol Fifteen micrograms of this DNase-treated RNA were reverse transcribed and amino allyl dUTP was incorporated in a reaction containing 500 ng oligo (dT) primers, 1x first strand buffer (Invitrogen, Carlsbad, CA), 0.01 M DTT, 500 µM each of dATP, dCTP, dGTP, and dTTP/aadUTP (2:3 ratio) 40 Units of rRNasin (Promega, Madison, WI) and 200Units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). After brief denaturation and annealing of the primers at 70˚C for 8 mins, the reaction was incubated at 42˚C for 2 hours, followed by alkali hydrolysis of RNA and cDNA purification using Microcon-30 columns (Millipore, Bedford, MA) according to the manufacturer’s instructions. The cDNA was then labeled with Cy5 by a coupling reaction using FluoroLink TM monofunctional dyes (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacture’s specifications. Probes were then purified using StrataPrep PCR Purification Kit (Stratagene, La Jolla, CA).
 
Channel 2
Source name GATA5 (-/-) RW-4 ES cells
Organism Mus musculus
Characteristics GATA5 (-/-) RW-4 ES cells, treated with retinoic acid
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from 60-80% confluent ES cell cultures. Total RNA was isolated using the guanidinium/isothiocyanate/phenol/chloroform method as previously described (Schultz). Total RNA was DNase treated using “DNA free” kit (Ambion, Austin, TX) according to manufacturer’s specifications.
Label Cy3
Label protocol Fifteen micrograms of this DNase-treated RNA were reverse transcribed and amino allyl dUTP was incorporated in a reaction containing 500 ng oligo (dT) primers, 1x first strand buffer (Invitrogen, Carlsbad, CA), 0.01 M DTT, 500 µM each of dATP, dCTP, dGTP, and dTTP/aadUTP (2:3 ratio) 40 Units of rRNasin (Promega, Madison, WI) and 200Units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). After brief denaturation and annealing of the primers at 70˚C for 8 mins, the reaction was incubated at 42˚C for 2 hours, followed by alkali hydrolysis of RNA and cDNA purification using Microcon-30 columns (Millipore, Bedford, MA) according to the manufacturer’s instructions. The cDNA was then labeled with Cy3 by a coupling reaction using FluoroLink TM monofunctional dyes (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacture’s specifications. Probes were then purified using StrataPrep PCR Purification Kit (Stratagene, La Jolla, CA).
 
 
Hybridization protocol Hybridization to microarrays was performed overnight at 42°C in hybridization buffer (25% formamide, 5 x SSC, 0.1% SDS, and 100 ug/ml sonicated salmon sperm DNA). After hybridization, the arrays were washed two times in washing buffer A (2 x SSC, 0.1% SDS) at 42°C for 5 min and then three times in washing buffer B (0.1 x SSC, 0.1% SDS) at room temperature for 10 min. The slides were briefly dipped into distilled water and dried in a stream of nitrogen before scanning.
Scan protocol Images were obtained by scanning of the arrays in an Affymetrix 428 scanner.
Description PMT was set at 30 for Cy5, and 35 for Cy3.
Data processing Signal intensities for Cy3- and Cy5-labeled probes were extracted by ImaGene software package version 4.2 using default settings and auto image segmentation. Mean and median intensities for signal and background as well quality characteristics (“empty” or “poor”) of the spots were obtained at this time.
 
Submission date Oct 25, 2006
Last update date Oct 30, 2006
Contact name Xiangxi Xu
E-mail(s) Xiangxi.Xu@fccc.edu
URL http://www.fccc.edu
Organization name Fox Chase Cancer Center
Department Ovarian Cancer and Tumor Cell Biology
Street address 333 Cottman Ave
City Philadelphia
State/province PA
ZIP/Postal code 19111
Country USA
 
Platform ID GPL4486
Series (1)
GSE6133 Alteration of differentiation potentials by modulating GATA transcription factors in murine embryonic stem cells

Data table header descriptions
ID_REF
VALUE pair Mean ratio; Correct for Background Intensity using Local group median and clique size 5; Set values below 20.0 to 20.0; Normalization using method Piece-wise Linear and Use All Genes

Data table
ID_REF VALUE
FCCC_moNIA15k-1 0.1703
FCCC_moNIA15k-2 0.6796
FCCC_moNIA15k-3 0.9929
FCCC_moNIA15k-4 -0.3214
FCCC_moNIA15k-5 0.509
FCCC_moNIA15k-6 0.5432
FCCC_moNIA15k-7 0.312
FCCC_moNIA15k-8 -0.4252
FCCC_moNIA15k-9 0.2587
FCCC_moNIA15k-10 0.2031
FCCC_moNIA15k-11 -0.0047
FCCC_moNIA15k-12 -0.2312
FCCC_moNIA15k-13
FCCC_moNIA15k-14 0.83
FCCC_moNIA15k-15 -0.0241
FCCC_moNIA15k-16 0.927
FCCC_moNIA15k-17 0.2091
FCCC_moNIA15k-18 0.4424
FCCC_moNIA15k-19 0.0472
FCCC_moNIA15k-20

Total number of rows: 15552

Table truncated, full table size 390 Kbytes.




Supplementary file Size Download File type/resource
GSM142373_cy3.tif.gz 15.8 Mb (ftp)(http) TIFF
GSM142373_cy3.txt.gz 3.0 Mb (ftp)(http) TXT
GSM142373_cy5.tif.gz 15.3 Mb (ftp)(http) TIFF
GSM142373_cy5.txt.gz 3.0 Mb (ftp)(http) TXT

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