|
| Status |
Public on Jul 18, 2011 |
| Title |
6h Control replicate 3 repetition (IMPPC) |
| Sample type |
RNA |
| |
|
| Source name |
Cervical cancer cell line (HeLa)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
treatment: serum deprived for 24 hours, followed by culture without EGF addition into the medium for 6 additional hours
|
| Treatment protocol |
For treatments, the cells were transferred to 60 mm dishes and cultured for 48h in complete growth medium, after which they were starved for 24h in DMEM containing 2% FBS. After serum deprivation cells were stimulated with EGF (150 ng/ml) for the indicated times. Three biological replicate experiments were performed, each comparing EGF treated with untreated control HeLa cells.
|
| Growth protocol |
HeLa cells were cultured at 37ºC in a 95/5 Air/CO2 water saturated atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat inactivated foetal bovine serum (FBS), 2 mM L-glutamine and 100U/ml Penicilin/streptomycine (complete growth medium).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from HeLa cells was extracted using the RNeasy RNA Isolation kit (Qiagen) followed by treatment with RNase-free DNase I (Ambion, Austin, TX) following manufacturer's instructions. RNA quantification was performed with a Nanodrop spectrophotometer, and the RNA integrity was assessed using the Bioanalyzer 2100 nanoelectrophoresis Lab-on-a–chip system (Agilent, Wilmington, DE). All RNAs used had RIN (RNA integrity number) ranging between 9.3 and 10, and 28S/18S ratios between 1.67 and 2.07.
|
| Label |
biotin / streptavidin-phycoerythrin
|
| Label protocol |
5 µg of total RNA was converted into double-stranded cDNA using the Superscript II system for cDNA synthesis (Invitrogen). Double-stranded cDNA was purified and in vitro transcribed. Biotinylated cRNA was purified and fragmented randomly to an average length of ~100 nt by incubating at 94°C for 35 min.
|
| |
|
| Hybridization protocol |
Microarrays were hybridized with 15 µg of fragmented cRNA. Hybridizations were carried out at 45°C for 16 h without agitation. Arrays were washed and the hybridized RNA was fluorescence-stained by incubating with streptavidin–phycoerythrin.
|
| Scan protocol |
Detection and feature readout were performed using the CCD-based detection system of the Geniom device (Cy3 filter set).
|
| Description |
HeLa cells, serum deprived for 24 hours, followed by culture without EGF addition into the medium for 6 additional hours
6h time point, Control, biological replicate 3 of 3. Technical replicate 2 of 2.
|
| Data processing |
Extracted raw data were filtered and normalized using the Limma package developed within the Bioconductor project in the R statistical programming environment. The single channel intensity data were quantile normalized to scaling across chips. Differentially expressed genes for each time point were chosen using as cut off criteria a SAM FDR of 5% and an average absolute fold change (|FC|) above 1.2 .
|
| |
|
| Submission date |
Jul 29, 2009 |
| Last update date |
Jul 18, 2011 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
lsumoy@igtp.cat
|
| Organization name |
IGTP
|
| Department |
High Content Genomics and Bioinformatics
|
| Street address |
Ctra. Can Ruti, Camí de les escoles s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8948 |
| Series (1) |
| GSE17403 |
Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis |
|
