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Sample GSM497554 Query DataSets for GSM497554
Status Public on Jan 12, 2011
Title T3_MEF-1, biological rep1
Sample type RNA
 
Source name hES-T3 cells grown on MEF feeder
Organism Homo sapiens
Characteristics growth conditions: grown on MEF feeder
gender: female
cell line: hES-T3
Treatment protocol No
Growth protocol Human embryonic stem cell line hES-T3, which is one of the five hES cell lines derived in our laboratory, exhibits normal female karyotype (46, XX), and it has been continuously cultured on mitomycin C (10 ug/ml) mitotically inactivated MEF feeder in hES medium under 5% CO2 at 37oC and underwent freezing/thawing processes. The hES culture medium consisted of DMEM/F12 (1:1, GIBCO) supplemented with 20% KSR (Invitrogen), 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM £]-mercaptoethanol, and 4 ng/ml human basic fibroblast growth factor (bFGF; Life Technologies). Routine passages of hES-T3 cells every 5-7 days were done with collagenase (type IV, 1 mg/ml, Invitrogen) treatment and mechanical scrape. The cryopreserved stock of hES-T3 cells (36 passages) were continuously maintained on MEF feeder for additional 14 passages, and these the hES-T3 cells were designated as T3/MEF.
Extracted molecule total RNA
Extraction protocol Trizol extraction of total RNA was performed according to the manufacturer's instructions. Total RNAs extracted using TRIZOL reagent were quantified spectrophotometrically. The cDNA synthesis was carried out by using SUPERSCRIPTTM one-step RT-PCR kit (Invitrogen). For the reverse transcription step, the whole 5 ul of the resuspended RNAs were incubated for 60 min at 42oC, then 15 min at 72oC in 50 ul of reaction mixture containing 25 ul of 2x Reaction Mix (Invitrogen) and 1 ul of RT/PLANTINUM Taq Mix (Invitrogen). 28.5 ul of the cDNAs present in the 50 ul RT reaction mixture purified by Microarray Target Purification Kit (Roche Applied Science, Indianapolis, ID, USA, http://www.roche-applies-science.com) were used as templates to amplify cDNA by Microarray Target Amplification Kit. The amplified cDNAs were purified by Microarray Target Purification Kit, and complementary RNAs (cRNAs) were synthesized from 200 ng cDNA with Microarray RNA Target Synthesis Kit (Roche Applied Science).
Label biotin
Label protocol Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 micrograms purified cRNA (Expression Analysis Technical Manual, 2004, Affymetrix).
 
Hybridization protocol Following fragmentation, 10 micrograms of cRNA were hybridized for 16 hr at 45oC on Affymetrix Human Genome U133 plus 2.0 GeneChip. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol GeneChips from the hybridization experiments were read by the Affymetrix GeneChip scanner 3000.
Description The mRNA profiles from T3/CMHDF, T3/CMMEF, T3/HDF and T3/MEF were quantitatively determined.
Data processing The data were processed by GeneSpring software version 7.3 GC-RMA preprocessor to use on the CEL files
 
Submission date Jan 14, 2010
Last update date Jan 12, 2011
Contact name Sung-Liang Yu
E-mail(s) slyu@ntu.edu.tw
Phone 886-2-23958341
Organization name National Taiwan University
Department Clinical Laboratory Sciences and Medical Biotechnology
Lab Microarray Core Facility
Street address Jen Ai Road Section1
City Taipei
ZIP/Postal code 100
Country Taiwan
 
Platform ID GPL570
Series (1)
GSE19902 Comparative expression profiles of human embryonic stem cells among different feeders and conditioned media

Data table header descriptions
ID_REF Affymetrix Synthetic ID
VALUE GC-RMA-calculated Signal intensity

Data table
ID_REF VALUE
1007_s_at 0.921
1053_at 1.168
117_at 1.353
121_at 1.528
1255_g_at 31.21
1294_at 0.421
1316_at 2.142
1320_at 0.681
1405_i_at 1.015
1431_at 1.099
1438_at 0.718
1487_at 0.917
1494_f_at 1.003
1552256_a_at 0.597
1552257_a_at 1.368
1552258_at 1.051
1552261_at 1
1552263_at 0.894
1552264_a_at 0.933
1552266_at 1.231

Total number of rows: 54675

Table truncated, full table size 888 Kbytes.




Supplementary file Size Download File type/resource
GSM497554_T3_MEF_1.CEL.gz 4.2 Mb (ftp)(http) CEL
Processed data included within Sample table

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