|
| Status |
Public on Aug 06, 2012 |
| Title |
MCF7_siFos_vehicle_24h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
MCF7 cells_c-Fos knockdown_Vehicle treated
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: breast cancer cells
transfected with: pool of siRNAs targeting c-Fos for 72hr
treated with: vehicle for 24hr
|
| Treatment protocol |
MCF-7 cells were transfected with a control siRNA or with the pool of siRNAs targeting c-Fos. At 6h after transfection, the medium was changed into phenol red-free DMEM plus 2% charcoal-dextran-treated calf serum for 72 h and were then treated with vehicle or E2 for 24 h.
|
| Growth protocol |
The human breast cancer cell line MCF7 was maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37°C, under a humidified atmosphere of 5% carbon dioxide.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy Mini Kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the Low Input Quick Amp Labeling Kit, One-Color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >=6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K Microarrays (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray C Scanner using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
SiFos-EtOH_2
Gene expression after 24 h vehicle treatment in si_c-Fos treated cells
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (protocol GE1-1010_Sep10 and Grid: 028004_D_F_20100430) to obtain background subtracted and spatially detrended Processed Signal intensities. Data were subsequently subjected to quantile normalization in Partek Genomics Suite (6.5).
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| |
|
| Submission date |
Mar 17, 2012 |
| Last update date |
Aug 06, 2012 |
| Contact name |
Chunyan Zhao |
| E-mail(s) |
chunyan.zhao@ki.se
|
| Phone |
46-8-52481126
|
| Fax |
46-8-7745538
|
| Organization name |
Karolinska Institute
|
| Department |
Biosciences and Nutrition
|
| Street address |
Hälsovägen 7-9
|
| City |
Stockholm |
| ZIP/Postal code |
SE-171 77 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE36586 |
Interplay between AP-1 and ERalpha in regulating gene expression and proliferation networks in breast cancer cells |
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