|
| Status |
Public on Jun 11, 2013 |
| Title |
6h EGF replicate 3 versus pool of controls |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cervical cancer cell line (HeLa)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
agent: EGF
|
| 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 cultured without EGF addition into the medium or were stimulated with EGF (150 ng/ml) for 6 hours.
|
| 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 fetal 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 miRvana RNA Isolation kit (Ambion) 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 |
Hy3
|
| Label protocol |
Total RNA (0.5 µg) was 3'-end-labeled using T4 RNA ligase and a Cy3/Cy5-labeled RNA linker for 1 hour at 30°C, and terminated by incubation for 15 min at 65°C.
|
| |
|
| Channel 2 |
| Source name |
Cervical cancer cell line (HeLa)_pool of 3 control samples
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
agent: control
common reference composition: pool of three replicates
|
| 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 cultured without EGF addition into the medium or were stimulated with EGF (150 ng/ml) for 6 hours.
|
| 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 fetal 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 miRvana RNA Isolation kit (Ambion) 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 |
Hy5
|
| Label protocol |
Total RNA (0.5 µg) was 3'-end-labeled using T4 RNA ligase and a Cy3/Cy5-labeled RNA linker for 1 hour at 30°C, and terminated by incubation for 15 min at 65°C.
|
| |
|
| |
| Hybridization protocol |
Labelled RNAs (20 µL) were combined with Hybridisation Buffer and incubated 5 min at 95ºC. Samples were hybridized in a volume of 440 ul to the miRCURY LNA microRNA Array V9.2 (Exiqon) for 16–20 hours at 65°C and 15 rpm. Post-hybridization washes were in 4x SSC at 60°C to remove the coverslip, followed by three times in 2x SSC, 0.025% SDS for 5 min each, three times in 0.8x SSC for 2 min each, and two times in 0.4x SSC for 3 min each.
|
| Scan protocol |
Microarrays were scanned on a Imagene v7.0 software
|
| Description |
Sample name: 6h-EGF-3
6h time point, biological replicate 3 of 3. HeLa cells treated with EGF versus pool of 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 intensities were imported without background corection and the Hy3/Hy5 log2ratios were normalized using lowess and scaled between arrays by quantile method. Differentially expressed genes between the treated and control condition were chosen using as cut off criteria a SAM FDR of 5% and an average absolute fold change (|FC|) above 1.2 .
|
| |
|
| Submission date |
Sep 28, 2012 |
| Last update date |
Jun 11, 2013 |
| 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 |
GPL16118 |
| Series (2) |
| GSE41225 |
Multiple platform assessment and isomir analysis of the EGF dependent microRNA-ome by microarray and deep sequencing analysis [Exiqon] |
| GSE41360 |
Multiple platform assessment and isomir analysis of the EGF dependent microRNA-ome by microarray and deep sequencing analysis |
|
