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Sample GSM559566 Query DataSets for GSM559566
Status Public on Jun 24, 2010
Title SV40-immortalized_human_corneal_epithelial_cells_rep3
Sample type RNA
 
Source name SV40-immortalized human corneal epithelial cell line
Organism Homo sapiens
Characteristics cell line: SV40
teer: 468 Ω x cm2
Treatment protocol Standard culture protocol
Growth protocol iHCE cells (passages of 22–23) were cultured in DMEM/Ham’s F12 (1:1) (Gibco, Invitrogen, Paisley, U.K.), 15% FBS (Gibco, Invitrogen), 0.3 mg/ml L-glutamine (Gibco, Invitrogen), 5 µg/ml insulin (Gibco, Invitrogen), 0.1 µg/ml cholera toxin (Calbiochem, La Jolla, CA), 10 ng/ml EGF (Invitrogen, Carlsbad, CA), 0.5% dimethylsulfoxide (DMSO; Sigma, St. Louis, MO), 0.1 mg/ml streptomycin and 1000 IU/ml penicillin (Gibco, Invitrogen). Cells were seeded on collagen coated permeable inserts (Transwell® Polyester Membrane Insert, Costar, Cambridge, MA) and maintained submerged into medium. After one week in culture the medium was supplemented with 40 µg/ml L(+)-ascorbic acid (Sigma, St. Louis, MO) and cells were changed to grow at the air-liquid interface (medium in the apical side was removed). Trans-epithelial electrical resistance (TEER) was followed with an EVOM resistance meter in Endohm chambers (World Precision Instruments, Sarasota, FL).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using TriReagent (Sigma, St. Louis, MO) and RNA samples were further purified using RNAeasy spin columns (Qiagen, Valencia, CA). RNA concentration and purity were determined by measuring absorbances (260 nm and 280 nm) and integrity was determined using the Agilent 2100 BioChip (Agilent Technologies., Palo Alto, CA).
Label biotin
Label protocol Biotin labeling of cRNA was performed using the ENZO RNA transcript labeling Kit 9 (BioArray High Yeald Labeling Kit, P/N 900182). IVT cRNA cleanup and quantitation, cRNA fragmentation, control targets were performed according to Affymetrix protocols. cDNA was synthesized using T7-(dT)24 Primer (Genset Corp) and Superscript Choice System (Gibco BRL Life technologies).
 
Hybridization protocol The hybridization cocktail was made according to Affymetrix protocols. The cocktail was heated to 99°C and then kept on heat block for 5 mins at 45°C. The probe array was incubated with 1X hybridization buffer (MES, 1M [Na], 20mM EDTA, 0.01% Tween) for 10 mins at 45°C. Samples were hybridized for 16 h at 45°C using a final volume of 200 µl. GeneChip Fluidics Station 450 using fluidics protocol EukGE-WS2v4 was utilized for washing. Hybridized microarrays were stained with a streptavidin-phycoerythrin (SAPE) reagent.
Scan protocol Hybridized microarrays were stained with a streptavidin-phycoerythrin (SAPE) reagent and fluorescence images were captured using an Agilent G2500A laser scanner.
Description Sample comprises of RNA isolated from immortalized human corneal epithelial cells cultured on the collagen coated permeable support at the air-liquid interface.
Data processing The data were analyzed with R v. 2.8.0 Bioconductor. Probes present in HGU133A were re-annotated according to Entrez Gene database using custom CDF v. 10. RMA algorithm was used to calculate relative gene expression values.
 
Submission date Jun 23, 2010
Last update date Jun 23, 2010
Contact name Kati-Sisko Vellonen
E-mail(s) Kati-Sisko.Vellonen@helsinki.fi
Phone +358-9-19159520
Fax +358-9-19159725
Organization name University of Helsinki, Faculty of Pharmacy
Department Centre for Drug Research
Street address Viikinkaari 5 E (P.O.Box 56)
City Helsinki
ZIP/Postal code FIN-00014 University of Helsinki
Country Finland
 
Platform ID GPL96
Series (1)
GSE22539 Gene expression profile of the SV40-immortalized human corneal epithelial cells

Data table header descriptions
ID_REF
VALUE RMA

Data table
ID_REF VALUE
1007_s_at 9.410850986
1053_at 8.080729669
117_at 5.718108418
121_at 7.842291333
1255_g_at 4.059583147
1294_at 7.423924366
1316_at 5.050649749
1320_at 4.989101116
1405_i_at 4.539971407
1431_at 4.04041202
1438_at 6.283123732
1487_at 7.390656498
1494_f_at 5.626151492
1598_g_at 9.20445411
160020_at 6.929295317
1729_at 8.257293324
1773_at 5.359913679
177_at 5.913847759
179_at 7.773637607
1861_at 6.803082559

Total number of rows: 22283

Table truncated, full table size 496 Kbytes.




Supplementary file Size Download File type/resource
GSM559566.cel.gz 2.3 Mb (ftp)(http) CEL
Raw data provided as supplementary file
Processed data included within Sample table

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