|
| 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) |
[email protected], [email protected]
|
| 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 |
|
