|
| Status |
Public on Jul 18, 2011 |
| Title |
6h EGF versus Control replicate 3 dye swap (Agilent) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cervical cancer cell line (HeLa)
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: serum deprived for 24 hours, followed by exposure to EGF for 6 hours
cell line: HeLa
|
| 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.
|
| 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 |
Cy3
|
| Label protocol |
500 ng of total RNA were reverse transcribed, amplified, labelled by in vitro transcription using the Low Input Linear Amplification Kit (Agilent 5184-3523), and hybridized following the manufacturer’s instructions (SSC wash Agilent 60-mer oligo microarray processing protocol, Version 4.1, April 2004).
|
| |
|
| Channel 2 |
| Source name |
Cervical cancer cell line (HeLa)
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: serum deprived for 24 hours, followed by culture without EGF addition into the medium for 6 additional hours
cell line: HeLa
|
| 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.
|
| 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 |
Cy5
|
| Label protocol |
500 ng of total RNA were reverse transcribed, amplified, labelled by in vitro transcription using the Low Input Linear Amplification Kit (Agilent 5184-3523), and hybridized following the manufacturer’s instructions (SSC wash Agilent 60-mer oligo microarray processing protocol, Version 4.1, April 2004).
|
| |
|
| |
| Hybridization protocol |
Three biological replicate experiments were performed, each comparing EGF treated with untreated control HeLa cells. Each experimental pair of labelled samples was co-hybridized on two separate microarrays with dye swapping to correct for dye bias effects. Thus, six microarray hybridizations were processed for each of three biological replicates for the two time points, totalling 12 array datasets.
|
| Scan protocol |
Fluorescent images were obtained using an Agilent G2565BA scanner at 100% PMT 100% laser power settings and quantified through the GenePix 6.0 software (Axon, Molecular Devices, Sunnywale, CA) using the irregular feature finding option.
|
| Description |
6h time point, biological replicate 3 of 3. Technical replicate 2 of 2. HeLa cells treated with EGF compared to untreated controls.
|
| 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 two channels were balanced by lowess normalization using 0.3 as the span parameter with reduced weights in control and poor quality spots, followed by 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 |
GPL4133 |
| Series (1) |
| GSE17403 |
Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis |
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