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Sample GSM81955 Query DataSets for GSM81955
Status Public on Jan 01, 2006
Title INSENS 5 4hr
Sample type RNA
 
Channel 1
Source name T6.01
Organism Homo sapiens
Characteristics lymphoblastoid cell line from T6.01, one out of seven of insensitive group, 50*10e6 cells
Biomaterial provider established in department of Otolaryngology / Head-Neck Surgery, VUmc, Amsterdam, the Netherlands
Treatment protocol bleomycin 4hr 5mU/ml
Extracted molecule total RNA
Label Cy3
Label protocol Non-amplified total RNA (75 µg) was used for preparation of labelled cDNA essentially as described (Buermans HP, et al. Physiol Genomics 2005;21:314-23). In brief, anchored oligo-dT [(dT)20-VN] primer was used and SuperScript II Reverse Transcriptase (Invitrogen), with incorporation of amino-allyl dUTP(Ambion, Huntingdon, UK). RNA was hydrolysed with 0.1 mM NaOH (70°C, 10 min) and after neutralizing with 0.1 mM HCl the samples were diluted in water and transferred onto a Microcon YM-30 filter (Millipore, Billerica, MA, USA) to purify and concentrate the cDNA. Before labelling with the dye, the dry cDNA pellet was dissolved in 9 ml of NaHCO3/NaCO3 (50 mM, pH 9.0) and incubated at room temperature for 1 hr in the dark with 2 ml aliquots Cy 3(Fluorolink Cy 3 Monofunctional Dye (Amersham). Unbound dye was removed using the Qiaquick PCR purification kit (Qiagen, Venlo, the Netherlands)according to the manufacturer’s protocol.
 
Channel 2
Source name Reference pool
Organism Homo sapiens
Characteristics Pooled RNA from a number of lymphoblastoid cell lines (with and without bleo treatment) and head and neck cancer cell lines.
Biomaterial provider established in department of Otolaryngology / Head-Neck Surgery, VUmc, Amsterdam, the Netherlands
Treatment protocol untreated
Extracted molecule total RNA
Label Cy5
Label protocol see label protocol of ch1 (Cy5 instead of Cy3)
 
 
Hybridization protocol According to Buermans HP, et al. Physiol Genomics 2005;21:314-23
Scan protocol According to Buermans HP, et al. Physiol Genomics 2005;21:314-23
Description Not applicable
Data processing ImaGene feature extraction (Biodiscovery Ltd, Marina del Rey, CA, USA) was used to record spot intensities. The signal mean was taken to represent the actual spot intensity, after subtraction of the mean background values. All spot were log2 transformed and intensities lower than 10, typical for the empty spots, were classified as missing. Next for each micro-array, the spot intensities were Z score normalized (Cheadle C, et al. J Mol Diagn 2003;5:73-81), while missing values (some 6% overall) were handled so as to minimize their effect on statistics. Specifically, such values were set equal to the mean of the micro-array distribution after Z score normalization, thus equal to zero. So for each micro-array, Z scores were computed for each green labeled spot intensity according to(2log(G) - mu) / sigma, where mu is the mean and sigma the standard deviation of all 2log(G) of the micro-array. Likewise the Z score was computed for each red labeled spot intensity and the result subtract from the ‘green’ Z score for each spot on the micro-array. These differences were then again Z score normalized to obtain the so called Z ratio for each spot.This information will soon be available in a publication.
 
Submission date Nov 04, 2005
Last update date Feb 12, 2007
Contact name Daoud Sie
E-mail(s) d.sie@vumc.nl
Phone +31 20 4442428
Organization name Vrije Universiteit Medical Center
Department Pathology
Lab Microarray Core Facility
Street address De Boelelaan 1117
City Amsterdam
ZIP/Postal code 1081 HV
Country Netherlands
 
Platform ID GPL2902
Series (1)
GSE3598 Bleomycin exposed lymphoblastoid cells

Data table header descriptions
ID_REF unique number of spot
CH1_MEAN Cy3 mean signal intensity
CH1BKD_MEAN
CH2_MEAN Cy5 mean signal intensity
CH2_BKD_MEAN Cy5 mean background intensity
VALUE The signal mean of each channel was taken to represent the actual spot intensity, after subtraction of the mean background values. Intensities lower than 10 were neglected and all values above 10 were log2 transformed .The spot intensities were Z score normalized (Cheadle C, et al. J Mol Diagn 2003;5:73-81), while missing values (some 6% overall) were handled so as to minimize their effect on statistics. Specifically, such values were set equal to the mean of the micro-array distribution after Z score normalization, thus equal to zero. So for each micro-array, Z scores were computed for each ch1 spot intensity according to(2log(G) - mu) / sigma, where mu is the mean and sigma the standard deviation of all 2log(G) of the micro-array. Likewise the Z score was computed for each ch2 labeled spot intensity and the result subtracted from the green Z score for each spot. These differences were then again Z score normalized to obtain the so called Z ratio for each spot.This Z-ratio defines VALUE.

Data table
ID_REF CH1_MEAN CH1BKD_MEAN CH2_MEAN CH2_BKD_MEAN VALUE
1 28632.62162 182.2489083 28066.57297 289.9650655 1.5600
2 339.5837838 194.3886463 1070.886486 290.8733624 -1.2300
3 381.8540541 167.5836576 1099.372973 276.9182879 -0.7630
4 28771.42162 188.9522059 29394.7027 316.5183824 1.5200
5 371.2242424 141.4709898 898.0121212 230.7440273 -0.4060
6 452.7272727 153.8860294 876.2121212 234.1875 0.0016
7 30660.45946 153.4752066 28565.29189 320.768595 1.6300
8 1543.85446 131.3275862 3367.755869 216.1293103 0.1420
9 300.741784 193.9036145 399.8497653 351.5361446 1.8200
10 28164.20541 285.8099174 27841.37297 541.4049587 1.5500
11 239.7307692 114.6496815 666.7769231 226.0636943 -0.7250
12 479.7276995 195.7521008 977.9765258 324.7184874 -0.0885
13 27902.15962 218.2136364 26023.15493 430.4409091 1.6300
14 757.9953052 260.9189189 2726.624413 418.4234234 -0.8910
15 1512.004695 155.7368421 2322.164319 362.9473684 0.6960
16 20612.26291 241.7981073 21909.53521 453.9495268 1.4200
17 358.6186441 192.4342105 568.7118644 377.2039474 0.7030
18 450.2484848 250.2337662 870.4545455 515.4588745 0.1900
19 24747.2723 335.4705882 23941.62441 545.6260504 1.5600
20 672.8591549 295.3629032 1201.57277 467.7822581 0.1570

Total number of rows: 19200

Table truncated, full table size 1119 Kbytes.




Supplementary data files not provided

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