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Sample GSM662820 Query DataSets for GSM662820
Status Public on Feb 02, 2012
Title G1 BaP-treated 12 h replicate 2
Sample type RNA
 
Channel 1
Source name G1 BaP-treated 12 h replicate 2
Organism Homo sapiens
Characteristics cell line: MCF-7
phase: G1
treatment: BaP for 12 hours
biological replicate: 2
Treatment protocol Cells were seeded at 2 × 10e5 cells/ml and BaP, dissolved in dimethylsulphoxide (DMSO), was added to give a final concentration of 2.5 µM for 12 hours. DMSO only was added to control cultures and its volume was kept at 0.3% of the total culture volume. Cells were harvested by trypsinisation followed by washing with PBS. Cell pellets were stored at -80ºC until further analysis.
Growth protocol MCF-7 Cells were grown as adherent monolayers and maintained in Dulbecco’s modified Eagle’s medium with GlutamaxTM I, 1000 mg/L D-glucose and sodium pyruvate (Invitrogen, Paisley, UK) and supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen) and 100 U/mL penicillin and 100 g/mL streptomycin (Sigma-–Aldrich, Dorset, UK). Cells were incubated in a humidified 5% CO2 atmosphere at 37oC and sub-cultured every 72 h when the cells were 80% confluent. Culture conditions were manipulated in order to generate (1) asynchronous cells (N) by allowing normal growth for 72 h; (2) G0/G1-phase enriched cultures by serum deprivation for 48 h; (3) S-phase-enriched cultures by serum deprivation for 48 h followed by 18 h growth in complete media; and (4) G2/M-phase-enriched cultures by treatment for 24 h with 1 µg/mL aphidicolin followed by 0.25 μM colchicine for 12 h.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from cells using the Qiagen RNeasy Mini Kit protocol (RNeasy Mini Handbook, Qiagen, UK)
Label Cy3
Label protocol Total RNA (4 µg) was reverse transcribed into cDNA by first incorporating a T7 oligo-dT-Promoter primer prior to generation of fluorescent cRNA (complimentary RNA) using the Agilent's Quick Amp Labeling kit (Agilent Technologies, UK). The method uses T7 RNA polymerase, which simultaneously amplifies target material and incorporates Cy3- or Cy5-labelled CTP. Following this, the labelled cRNA was purified using the Qiagen RNeasy Mini spin columns and quantified using the NanoDrop ND-1000 instrument (Thermo Scientific, USA).
 
Channel 2
Source name universal human reference RNA
Organism Homo sapiens
Characteristics reference: Stratagene universal human reference RNA (UHRR)
Treatment protocol Cells were seeded at 2 × 10e5 cells/ml and BaP, dissolved in dimethylsulphoxide (DMSO), was added to give a final concentration of 2.5 µM for 12 hours. DMSO only was added to control cultures and its volume was kept at 0.3% of the total culture volume. Cells were harvested by trypsinisation followed by washing with PBS. Cell pellets were stored at -80ºC until further analysis.
Growth protocol MCF-7 Cells were grown as adherent monolayers and maintained in Dulbecco’s modified Eagle’s medium with GlutamaxTM I, 1000 mg/L D-glucose and sodium pyruvate (Invitrogen, Paisley, UK) and supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen) and 100 U/mL penicillin and 100 g/mL streptomycin (Sigma-–Aldrich, Dorset, UK). Cells were incubated in a humidified 5% CO2 atmosphere at 37oC and sub-cultured every 72 h when the cells were 80% confluent. Culture conditions were manipulated in order to generate (1) asynchronous cells (N) by allowing normal growth for 72 h; (2) G0/G1-phase enriched cultures by serum deprivation for 48 h; (3) S-phase-enriched cultures by serum deprivation for 48 h followed by 18 h growth in complete media; and (4) G2/M-phase-enriched cultures by treatment for 24 h with 1 µg/mL aphidicolin followed by 0.25 μM colchicine for 12 h.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from cells using the Qiagen RNeasy Mini Kit protocol (RNeasy Mini Handbook, Qiagen, UK)
Label Cy5
Label protocol Total RNA (4 µg) was reverse transcribed into cDNA by first incorporating a T7 oligo-dT-Promoter primer prior to generation of fluorescent cRNA (complimentary RNA) using the Agilent's Quick Amp Labeling kit (Agilent Technologies, UK). The method uses T7 RNA polymerase, which simultaneously amplifies target material and incorporates Cy3- or Cy5-labelled CTP. Following this, the labelled cRNA was purified using the Qiagen RNeasy Mini spin columns and quantified using the NanoDrop ND-1000 instrument (Thermo Scientific, USA).
 
 
Hybridization protocol After quantification of labeled cRNA, The Cy3-labelled samples were mixed with an appropriate volume of Cy5-labelled reference for each assay, concentrated to less than 41.8µl and hybridised to the oligo microarrays using the Gene Expression Hybridization Kit (Agilent Technologies, UK). The hybridisation was carried out using Agilent SureHyb chambers for 17 hours in Rotisserie Hyb Oven set to 65ºC (Agilent Technologies, UK) and rotating at 10 rpm.
Scan protocol After hybridisation, the arrays were washed by Agilent Gene Expression Wash Buffer following the instructions. The arrays were scanned with a Axon 4000B scanner at wavelength 635nm and 532nm.
Description 251485030758_G1-BaP2
Data processing The images were analysed with BlueFuse software to extract expression values, then all data were loaded into GeneSpring V7.2 software. The data were LOWESS normalised and natural log-ratios were used for t-test and other analyses. Normalised ratios were used for fold-change analysis.
 
Submission date Jan 27, 2011
Last update date Feb 02, 2012
Contact name Ian Giddings
E-mail(s) ian.giddings@icr.ac.uk, daniel.brewer@icr.ac.uk
Phone +44 20 8722 4293
Fax +44 20 8722 4141
URL http://www.crukdmf.icr.ac.uk
Organization name Institute of Cancer Research
Department Section of Molecular Carcinogenesis
Lab CANCER RESEARCH UK DNA Microarray Facility
Street address 15 Cotswold Road
City Sutton
State/province Surrey
ZIP/Postal code SM2 5NG
Country United Kingdom
 
Platform ID GPL6480
Series (1)
GSE26917 Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene

Data table header descriptions
ID_REF
VALUE Normalized natural log ratio (Cy3/Cy5) representing test/reference

Data table
ID_REF VALUE
A_23_P253586 -0.86059666
A_23_P217507 -0.14248356
A_24_P538590 0.8018343
A_24_P569294 0.3364249
A_23_P259451 0.10707635
A_32_P219520 -0.61378723
A_32_P38619 0.21591756
A_24_P153234 0.02682364
A_23_P76006 0.54090774
A_23_P381332 0.40914413
A_23_P83498 -1.4917339
A_32_P81149 -0.20214051
A_23_P413224 -2.4591346
A_23_P253597 0.059768755
A_24_P53985 0.1400967
A_23_P6321 -0.27849436
A_24_P390793 -0.449248
A_24_P640261 -2.5556638
A_23_P146885 -0.70266473
A_24_P170365 0.49017328

Total number of rows: 41094

Table truncated, full table size 930 Kbytes.




Supplementary file Size Download File type/resource
GSM662820.txt.gz 3.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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