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Sample GSM255691 Query DataSets for GSM255691
Status Public on Feb 29, 2008
Title CaCo2_TNF_60min_rep7
Sample type RNA
 
Source name CaCo-2 cell line
Organism Homo sapiens
Characteristics epithelial adherent cell line established from a human colon adenocarcinoma
Biomaterial provider German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany
Treatment protocol Cells were stimulated for 60 min with recombinant human TNF-α in a final concentration of 5 ng/ml.
Growth protocol The cells were cultured in DMEM containing 20% foetal bovine serum, 100 U/ml penicillin and 100 ng/ml streptomycin. Before passaging, the adherent cells were rinsed with PBS (37°C) three times to remove the fetal bovine serum that inhibits the trypsinization and trypsinized with 5-8 ml of Trypsine/EDTA solution at 37°C in the incubator. Confluent cell cultures (3-6 x 105 cells/cm2) were split, and seeded out for stimulation at 1 x 105 cells/cm2.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with minor modifications that improved RNA yields and purity in our hands. Cells were lysed in an appropriate volume of Buffer RLT. To facilitate complete lysis, samples were allowed to stand at room temperature for 5-10 min before vortexing. The lysate was homogenized by addition to a QIA shredder column (Qiagen) and centrifuged at 14,000 rpm for 2 min. The resulting supernatant was centrifuged at 14,000 rpm for 3 min to pellet any cell debris. One volume of 70% ethanol was added to the cleared lysate, added to the RNeasy column and centrifuged at 10,000 rpm for 15 sec. Bound RNA was washed with the addition of RW1 buffer to the column and allowed to stand at room temperature for 10 min followed by centrifugation at 10,000 rpm for 15 sec. DNase 1 incubation mix (Stock: 273 kunits; RNase-free DNase Set, Qiagen) was added directly to the spin column membrane and digestion of any contaminating genomic DNA was carried out at room temperature for 30 min. The column was washed with RWI buffer and the DNase treatment step was repeated. The bound RNA was washed with RW1 buffer and centrifuged at 10,000 rpm for 15 sec three times, followed by two additional wash and centrifugation steps with RPE buffer. Finally, purified RNA was eluted off the column with RNase-free water. RNA concentration and purity was determined spectrophotometrically at A260/A280 and was determined to be intact as assessed by formaldehyde gel electrophoresis. The presence of genomic DNA contamination was assessed by PCR amplification of GAPDH using standard PCR conditions and the following primers: GAPDH_F2-5’- ACCCACTCCTCCACCTTTGAC-3’, GAPDH_R2- 5’-CTGTTGCTGTAGCCAAATTCGT-3’. RNA was re-treated with DNase if necessary. Upon confirming the quality of the RNA (intact and free of genomic DNA), 15 µg of total RNA for expression screening.
Label α33P-dCTP random hexamer labeling
Label protocol 200 - 500 ng mRNA was isolated from total RNA sample using the Oligotex kit (Qiagen) according to the manufacturer’s instructions. Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983). Reversed transcribed cDNA were prepared in a final volume of 30microL as described in detail previously (labeling reaction at 42°C for 60 min; Eickhoff et al. 2000). RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl. Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
 
Hybridization protocol Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C. These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction. Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm). The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL). The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C. Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C. Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.
Scan protocol Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan).
Description Comparison of gene expression patterns of TNF-alpha stimulated and not stimulated CaCo-2 cells
Data processing The image data files were gridded using AIDA software (Raytest, Straubenhardt, Germany) and the results were imported in a custom-made database. The ranked expression data distribution was found to follow Zipf´s law (Hoyle et al, 2002) and the logarithmic normal transformed datasets were normalised following this principle, as previously described by Lu et al. 2005. Any hybridisation signal that had a value that fell below a predefined threshold (mean background + 2x standard deviation) were considered as indistinguishable from background and was set to zero. Those values where duplicate expression values for the same gene were not similar (outliers or false positive, difference between duplicates >20%) were eliminated.
 
Submission date Jan 08, 2008
Last update date Jan 14, 2008
Contact name Hermann Schulze
E-mail(s) schulzekiel@gmx.de
Organization name Universitätsklinik Rostock
Street address Ernst-Heydemann-Str. 6
City Rostock
ZIP/Postal code 18057
Country Germany
 
Platform ID GPL284
Series (1)
GSE10108 Gene expression analysis of TNF-alpha stimulated CaCo-2 cells

Data table header descriptions
ID_REF
SIGNAL_RAW1 weighted mean pixel intensity of first duplicate spot
SIGNAL_RAW2 weighted mean pixel intensity of second duplicate spot
VALUE final value after normalization and background substraction, "-1" indicates values omitted from further analysis due to bad duplicate or low signal intensity

Data table
ID_REF SIGNAL_RAW1 SIGNAL_RAW2 VALUE
01A01 8.5 6.7 0.888719723
01A02 4.8 5.8 0.572743461
01A03 6.4 7.1 0.65412204
01A04 6.7 5.4 0.737230837
01A05 5.9 4.8 0.464249685
01A06 4.9 5.8 0.496602297
01A07 7.6 9.4 0.70729585
01A08 5.6 6.1 0.476615362
01A09 5.2 5.2 0.480935596
01A10 5.3 6.5 0.662827998
01A11 4.1 5.1 0.493083686
01A12 12.1 11.8 2.438798368
01A13 4.6 5 0.609213606
01A14 5.2 5.8 0.513287142
01A15 5 4.9 0.54789415
01A16 4 5.3 0.4569363
01A17 46.7 48.1 8.377459526
01A18 5.1 5.7 0.519973561
01A19 5.6 5.9 0.604632512
01A20 6.2 6 0.682002425

Total number of rows: 34560

Table truncated, full table size 782 Kbytes.




Supplementary data files not provided
Processed data included within Sample table
Raw data included within Sample table

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