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Sample GSM29609 Query DataSets for GSM29609
Status Public on Aug 11, 2005
Title Crohn 04, CD04
Sample type RNA
 
Source name sigmoid colon biopsy
Organism Homo sapiens
Extracted molecule total RNA
 
Description Pathohistological and clinical examination of Patient CD04 (32 year-old female) yielded to diagnosis of Crohn's disease.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient 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 from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 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).
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. 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). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible.
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum. Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).
 
Submission date Aug 26, 2004
Last update date Oct 28, 2005
Contact name Robert Haesler
E-mail(s) r.haesler@mucosa.de
Phone +49 (0) 431 597 5754
Organization name University Clinic of Schleswig Holstein
Department IKMB / 1st Dept. of Medicine
Lab Functional Genomics Platform
Street address Schittenhelmstr. 12
City Kiel
ZIP/Postal code 24105
Country Germany
 
Platform ID GPL284
Series (1)
GSE1710 Expression profiling in inflammatory bowel disease

Data table header descriptions
ID_REF
SIGNAL_RAW weighted mean pixel intensity, mean of two spot replicates
VALUE final value after normalization and background subtraction, emtpy spots and spots below background were set to zero

Data table
ID_REF SIGNAL_RAW VALUE
01A01 0.05955 0.110732763367078
01A02 0.036 5.69176743670783E-02
01A03 0.02465 4.67664063670783E-02
01A04 0.0301 5.08064243670783E-02
01A05 0.028 4.45237973670783E-02
01A06 0.02895 4.72706023670783E-02
01A07 0.02295 4.46361223670783E-02
01A08 0.03135 4.87660753670783E-02
01A09 0.0244 3.86017033670783E-02
01A10 0.0312 4.71934663670783E-02
01A11 0.02625 4.01170243670783E-02
01A12 0.0344 5.77267923670783E-02
01A13 0.02395 3.82924813670783E-02
01A14 0.02535 3.09114223670783E-02
01A15 0.0259 4.52009653670783E-02
01A16 0.04245 6.35543013670783E-02
01A17 0.27165 0.304538576367078
01A18 0.02735 3.75579963670783E-02
01A19 0.026 4.44140203670783E-02
01A20 0.0264 4.41935723670783E-02

Total number of rows: 34560

Table truncated, full table size 1055 Kbytes.




Supplementary data files not provided

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