|
| Status |
Public on Nov 28, 2013 |
| Title |
WWOX–silenced subline B, replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MCF10 shWWOX-B
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10F mammary epithelial cells
treatment: WWOX–silenced subline B
|
| Treatment protocol |
Cells were infected with the following shRNA-expressing GIPZ lentiviruses (Open Biosystems) at an MOI of 5: Scrambled control shRNA (#RHS4348), shWWOX-A (#V2LHS_255213 ) or shWWOX-B (#V2LHS_255229). Co-infection of shWWOX-A and shWWOX-B was indicated as “shWWOX”. Cells were infected according to manufacturer’s instructions. Cells were selected for three days with 2 μg/ml puromycin and WWOX protein level was assayed by western blot.
|
| Growth protocol |
MCF10 cells were cultured in DMEM/F12 supplemented with 5% fetal bovine serum, 100 μg/mL hydrocortisone, 10 μg/mL insulin, 20 ng/mL EGF, 1 ng/mL cholera toxin and 1% penicillin-streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 3 biological replicates each of MCF10 Scr, MCF10 shWWOX-A and MCF10 shWWOX-B using the RNeasy Mini kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
2 µg of total RNA from each of WWOX–silenced sublines labeled with Cy5 were individually hybridized on Agilent Whole Human Genome 4X44K microarrays to analyze ~40000 transcripts (Agilent Technologies, Palo Alto, CA, USA) using the RNA derived from the corresponding MCF10 Scr sample (labeled with Cy3) as reference. For RNA labeling, we used the Quick Amp Kit (Agilent Technologies, Palo Alto, CA) by following the manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
MCF10 scrambled control shRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10F mammary epithelial cells
treatment: scrambled control shRNA
|
| Treatment protocol |
Cells were infected with the following shRNA-expressing GIPZ lentiviruses (Open Biosystems) at an MOI of 5: Scrambled control shRNA (#RHS4348), shWWOX-A (#V2LHS_255213 ) or shWWOX-B (#V2LHS_255229). Co-infection of shWWOX-A and shWWOX-B was indicated as “shWWOX”. Cells were infected according to manufacturer’s instructions. Cells were selected for three days with 2 μg/ml puromycin and WWOX protein level was assayed by western blot.
|
| Growth protocol |
MCF10 cells were cultured in DMEM/F12 supplemented with 5% fetal bovine serum, 100 μg/mL hydrocortisone, 10 μg/mL insulin, 20 ng/mL EGF, 1 ng/mL cholera toxin and 1% penicillin-streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 3 biological replicates each of MCF10 Scr, MCF10 shWWOX-A and MCF10 shWWOX-B using the RNeasy Mini kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
2 µg of total RNA from each of WWOX–silenced sublines labeled with Cy5 were individually hybridized on Agilent Whole Human Genome 4X44K microarrays to analyze ~40000 transcripts (Agilent Technologies, Palo Alto, CA, USA) using the RNA derived from the corresponding MCF10 Scr sample (labeled with Cy3) as reference. For RNA labeling, we used the Quick Amp Kit (Agilent Technologies, Palo Alto, CA) by following the manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
The hybridization steps were carried out according to the Agilent protocol.
|
| Scan protocol |
Scanned on a Genepix 4000B microarray scanner (Axon Instruments, Foster City, CA, USA).
|
| Description |
Biological replicate 1 of 3, WWOX–silenced subline B.
|
| Data processing |
Image analysis and initial quality control were performed using Agilent Feature Extraction Software v10.2.
We used the limma Bioconductor package for background adjustment (normexp method), within (Loess algorithm) and between (quantiles method) arrays normalization.
|
| |
|
| Submission date |
May 24, 2013 |
| Last update date |
Nov 28, 2013 |
| Contact name |
C. Marcelo Aldaz |
| E-mail(s) |
maaldaz@mdanderson.org
|
| Phone |
512-237-9530
|
| Organization name |
The University of TX M.D. Anderson Cancer Center
|
| Department |
Molecular Carcinogenesis
|
| Lab |
Aldaz Lab
|
| Street address |
POB 389
|
| City |
Smithville |
| ZIP/Postal code |
78957 |
| Country |
USA |
| |
|
| Platform ID |
GPL10332 |
| Series (1) |
| GSE47371 |
WWOX interacts with SMAD3 and modulates its transcriptional activity in breast cells |
|
