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Sample GSM249310 Query DataSets for GSM249310
Status Public on Jun 01, 2008
Title MCF7 P38 (2) X MCF7 DRA (2)
Sample type RNA
 
Channel 1
Source name MCF7 P38
Organism Homo sapiens
Characteristics Human breast cancer cell line
Extracted molecule total RNA
Extraction protocol Cells (70-80% confluent) cultured in 75 cm2 flasks were harvested using 1 ml/10 cm2 of TRIzol reagent (Invitrogen) and stored at -80ºC until use. RNA and DNA were extracted using TRIzol reagent and according to the manufacturers protocols. Nucleic acids were quantified using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA) and quality was visually confirmed by gel electrophoresis.
Label Cy3
Label protocol cDNA probes were generated from 30μg of total RNA, with an oligo(dT) [(dT)20-VN] primer (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with incorporation of dUTP, and indirectly labeled with cyanine-5 dCTP and cyanine-3 dCTP (Perkin-Elmer). cDNA was incubated at room temperature for 1 hour with fluorolink monofunctional Cy5 or Cy3 dye (Amersham, Roosendaal, The Netherlands) followed by 15 minutes of 4 mol/L hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen, Leusden, The Netherlands).
 
Channel 2
Source name MCF7 DRA
Organism Homo sapiens
Characteristics Human breast cancer cell line resistant to Vitamin D
Extracted molecule total RNA
Extraction protocol Cells (70-80% confluent) cultured in 75 cm2 flasks were harvested using 1 ml/10 cm2 of TRIzol reagent (Invitrogen) and stored at -80ºC until use. RNA and DNA were extracted using TRIzol reagent and according to the manufacturers protocols. Nucleic acids were quantified using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA) and quality was visually confirmed by gel electrophoresis.
Label Cy5
Label protocol cDNA probes were generated from 30μg of total RNA, with an oligo(dT) [(dT)20-VN] primer (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with incorporation of dUTP, and indirectly labeled with cyanine-5 dCTP and cyanine-3 dCTP (Perkin-Elmer). cDNA was incubated at room temperature for 1 hour with fluorolink monofunctional Cy5 or Cy3 dye (Amersham, Roosendaal, The Netherlands) followed by 15 minutes of 4 mol/L hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen, Leusden, The Netherlands).
 
 
Hybridization protocol cDNA was incubated at room temperature for 1 hour with fluorolink monofunctional Cy5 or Cy3 dye (Amersham, Roosendaal, The Netherlands) followed by 15 minutes of 4 mol/L hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen, Leusden, The Netherlands) and mixed with 12 µg poly(dA) (Amersham), 60 µg yeast tRNA (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands), and 24 µg Cot-1 DNA (Invitrogen). The labeled target was dissolved in 127-µL hybridization mixture containing 46% formamide (Invitrogen), 9.5% dextran sulfate (U.S. Biochemical Corp., Cleveland, USA), 2x SSC, and 0.2% SDS. The labeled target was heated to 70°C for 10 minutes and annealed at 37°C for 1 hour. Slides were hybridized at 37°C overnight (14h). After hybridization, the slides were washed in the HybArray 12, with 50% formamide (Fluka, Sigma-Aldrich Chemie), 2x SSC (pH 7) at 35°C for 15 minutes followed by PI buffer (0.1 mol/L sodium phosphate, 0.1% Igepal Ca630 (pH 8)) at room temperature and three washes of 0.2x SSC, 0.1x SSC, and 0.01x SSC at room temperature followed by centrifugation.
Scan protocol Microarray Scanner G2505B (Agilent Technologies), default settings
Description see Costa et all for additional information
Data processing Spot analysis and quality control were fully automated using BlueFuse version 3.2 (BlueGnome, Cambridge, UK) and spots with quality flag <1 or confidence value <0.1 were excluded from further analysis. After scanning and data acquisition, genes with spots flagged in more than one array were excluded from further analysis. For the remaining genes, the value of a flagged spot was imputed using K nearest neighbors’ imputation as implemented in the R-package “impute”. Curvature in the MA plots was limited but nevertheless normalized using lowess as implemented in the R-package “MAAnova”.
 
Submission date Dec 13, 2007
Last update date Dec 13, 2007
Contact name Daoud Sie
E-mail(s) [email protected]
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 GPL2826
Series (1)
GSE9867 Understanding Vitamin D resistance using expression microarrays

Data table header descriptions
ID_REF
AMPL_CY3 Total signal in channel 1 (Cy3)
AMPL_CY5 Total signal in channel 2 (Cy5)
FLAG 0-value not flagged, 1-value flagged in one array (IMPUTED), 2-value flagged in more than one array (NOT USED), 3-value flagged due to quality/confidence (NOT USED)
IMPUTE_CY3 Cy3 imputed value using the R-package “impute”
IMPUTE_CY5 Cy5 imputed value using the R-package “impute”
LOWESS_CY3 Lowess normalized Cy3 value
LOWESS_CY5 Lowess normalized Cy5 value
VALUE -[INV_VALUE]
INV_VALUE Log, base 2, of the ratio of the Lowess normalized Cy3 value divided by Lowess normalized Cy5 value

Data table
ID_REF AMPL_CY3 AMPL_CY5 FLAG IMPUTE_CY3 IMPUTE_CY5 LOWESS_CY3 LOWESS_CY5 VALUE INV_VALUE
1 221.214 254.806 2 null null null null null null
2 235.196 371.745 2 null null null null null null
3 718.944 1248.408 2 null null null null null null
4 1255.69 2041.376 2 null null null null null null
5 636.256 904.825 2 null null null null null null
6 287.076 467.411 2 null null null null null null
7 557.901 829.512 0 557.901 829.512 9.348591863 9.471392506 -0.122801 0.122800643
8 6429.845 6373.071 0 6429.845 6373.071 12.89559561 12.39274565 0.50285 -0.50284996
9 391.792 434.978 0 391.792 434.978 8.81926269 8.559480064 0.259783 -0.259782626
10 1232.006 1720.877 0 1232.006 1720.877 10.49737721 10.51834462 -0.0209674 0.020967414
11 215.311 283.043 2 null null null null null null
12 468.339 696.407 0 468.339 696.407 9.091668405 9.22352786 -0.131859 0.131859455
13 186.845 221.309 3 null null null null null null
14 5170.425 6690.954 0 5170.425 6690.954 12.57990525 12.46415811 0.115747 -0.115747139
15 628.717 927.52 2 null null null null null null
16 614.563 978.769 2 null null null null null null
17 1485.875 1896.567 2 null null null null null null
18 1180.288 971.978 0 1180.288 971.978 10.42586209 9.703840975 0.722021 -0.722021116
19 337.236 290.129 2 null null null null null null
20 244.039 256.809 0 244.039 256.809 8.111926678 7.823593189 0.288333 -0.288333489

Total number of rows: 30000

Table truncated, full table size 1964 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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