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Sample GSM5996 Query DataSets for GSM5996
Status Public on Dec 14, 2003
Title Patient C
Sample type RNA
 
Source name sigmoid colon biopsy
Organism Homo sapiens
Extracted molecule total RNA
 
Description Pathohistological and clinical examination of Patient C (62 year-old female) yielded no significant pathological findings. This patient was considered as a normal control for the purposes of the study.
A complete description of the RNA extraction is available under GSE405.

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. For the purposes of the microarray comparison study (GSM5994-5998), all filters were chosen from the same spotting production run and all filters were used for the first time.

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. The following output values from VisualGrid were used for subsequent data analysis: SIGNAL_RAW1 (the weighted mean pixel intensity within the node mask for the first duplicate), SIGNAL_RAW2 (the weighted mean pixel intensity within the node mask for the second duplicate), BKD_MEAN (the mean of the weighted means of all background spots in a 6 X 6 block). For this dataset (GSM5994-5998), the weight of each pixel was set to one.

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 Apr 15, 2003
Last update date Oct 28, 2005
Contact name Nancy Mah
E-mail(s) n.mah@mucosa.de
Phone +0049-431-597-3725
Organization name Christian-Albrechts-University Kiel
Street address
City Kiel
ZIP/Postal code D-24105
Country Germany
 
Platform ID GPL284
Series (1)
GSE405 Microarray Comparison Using Normal Human Colonic Mucosa

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
BKD_MEAN mean of the weighted means of all background spots in a 6X6 block
VALUE final value after normalization and background subtraction; values of -1 are values that have been omitted from analysis due to a bad duplicate or due to one raw signal being below the median filter background

Data table
ID_REF SIGNAL_RAW1 SIGNAL_RAW2 BKD_MEAN VALUE
01A01 0.060699999 0.060600001 0.037900001 0.056297
01A02 0.048300002 0.039700001 0.044199999 0.052044
01A03 0.036899999 0.034400001 0.043900002 0.035917
01A04 0.0495 0.043000001 0.0414 0.05574
01A05 0.087499999 0.054400001 0.090899996 0.054562
01A06 0.038800001 0.0359 0.045200001 0.038121
01A07 0.047800001 0.039900001 0.038199998 0.04921
01A08 0.041900001 0.037099998 0.044 0.037968
01A09 0.0416 0.039000001 0.046100002 0.037598
01A10 0.037 0.034200002 0.042300001 0.043629
01A11 0.037300002 0.032000002 0.043299999 0.031186
01A12 0.064199999 0.053399999 0.0491 0.070775
01A13 0.034200002 0.028999999 0.033500001 0.031493
01A14 0.035700001 0.029100001 0.0383 0.029592
01A15 0.034200002 0.0339 0.034699999 0.032589
01A16 0.028200001 0.026699999 0.034499999 0.032365
01A17 0.121100001 0.058800001 0.087200001 0.088502
01A18 0.066100001 0.0305 0.029200001 0.027982
01A19 0.046300001 0.036499999 0.0286 0.044797
01A20 0.0295 0.0274 0.029999999 0.030195

Total number of rows: 34560

Table truncated, full table size 1588 Kbytes.




Supplementary data files not provided

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