|
| Status |
Public on Dec 08, 2016 |
| Title |
PE5 (Cy5) Control (Cy3) - OVCAR8 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PE5-treated OVCAR8 cells. Biological replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OVCAR8
cell type: ovarian cancer cells
treatment: PE5
|
| Treatment protocol |
NCI/ADR-RES cells (2 x 105 per well) and OVCAR8 cells (5 x 104 per well) were seeded into 6-well plates and treated for 36 h with concentrations of PE5 or onconase that caused a 10% decrease of cell proliferation (12 µM PE5 or 0.5 µM onconase for NCI/ADR-RES cells and 0.45 µM PE5 or 0.06 µM onconase for OVCAR8 cells). Four independent biological replicates were performed for each cell line.
|
| Growth protocol |
NCI/ADR-RES cells were grown at 37°C in a humidified atmosphere of 5% CO2 in DMEM (Gibco, Germany) supplemented with 10% fetal bovine serum (Gibco, Germany), 50 U/ml penicillin, 50 μg/ml streptomycin (Gibco, Germany), and 1.84 μM doxorubicin (Tedec-Meijic Farma, Spain). OVCAR8 cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in RPMI (Gibco, Germany) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells remained free of Mycoplasma and were propagated according to established protocols.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After treatment, cells were harvested at 400 xg for 5 min at 4ºC and washed twice with cold PBS. Total RNA was extracted using the mirVana miRNA isolation kit (Applied Biosystems/Ambion, USA) according to the manufacturer’s instructions. RNA was then evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and a ND-1000 Spectrophotometer (NanoDrop) (Thermo Fisher Scientific, USA).
|
| Label |
Cy5
|
| Label protocol |
Sample preparation and microarray processing procedures were done according to the Two-Color Microarray-Based Gene Expression Analysis v. 6.5 (Agilent Technologies, USA). Briefly, 200 ng of total RNA was used to synthesize double-stranded cDNA with AffinityScript-Reverse Transcriptase and Oligo dT-Promoter Primer. cDNA was simultaneously amplified and transcribed into cyanine 3- or cyanine 5-labeled cRNA employing T7 RNA Polymerase in presence of cyanine 3-CTP or cyanine 5-CTP. Labeled cRNA (antisense) was then purified and evaluated using a ND-1000 Spectrophotometer (NanoDrop) (Thermo Fisher Scientific, USA).
|
| |
|
| Channel 2 |
| Source name |
Control OVCAR8 cells. Biological replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OVCAR8
cell type: ovarian cancer cells
treatment: Control (untreated)
|
| Treatment protocol |
NCI/ADR-RES cells (2 x 105 per well) and OVCAR8 cells (5 x 104 per well) were seeded into 6-well plates and treated for 36 h with concentrations of PE5 or onconase that caused a 10% decrease of cell proliferation (12 µM PE5 or 0.5 µM onconase for NCI/ADR-RES cells and 0.45 µM PE5 or 0.06 µM onconase for OVCAR8 cells). Four independent biological replicates were performed for each cell line.
|
| Growth protocol |
NCI/ADR-RES cells were grown at 37°C in a humidified atmosphere of 5% CO2 in DMEM (Gibco, Germany) supplemented with 10% fetal bovine serum (Gibco, Germany), 50 U/ml penicillin, 50 μg/ml streptomycin (Gibco, Germany), and 1.84 μM doxorubicin (Tedec-Meijic Farma, Spain). OVCAR8 cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in RPMI (Gibco, Germany) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells remained free of Mycoplasma and were propagated according to established protocols.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After treatment, cells were harvested at 400 xg for 5 min at 4ºC and washed twice with cold PBS. Total RNA was extracted using the mirVana miRNA isolation kit (Applied Biosystems/Ambion, USA) according to the manufacturer’s instructions. RNA was then evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and a ND-1000 Spectrophotometer (NanoDrop) (Thermo Fisher Scientific, USA).
|
| Label |
Cy3
|
| Label protocol |
Sample preparation and microarray processing procedures were done according to the Two-Color Microarray-Based Gene Expression Analysis v. 6.5 (Agilent Technologies, USA). Briefly, 200 ng of total RNA was used to synthesize double-stranded cDNA with AffinityScript-Reverse Transcriptase and Oligo dT-Promoter Primer. cDNA was simultaneously amplified and transcribed into cyanine 3- or cyanine 5-labeled cRNA employing T7 RNA Polymerase in presence of cyanine 3-CTP or cyanine 5-CTP. Labeled cRNA (antisense) was then purified and evaluated using a ND-1000 Spectrophotometer (NanoDrop) (Thermo Fisher Scientific, USA).
|
| |
|
| |
| Hybridization protocol |
Labeled cRNA (antisense) was hybridized to the oligonucleotide microarray at 65ºC for 17 h. After hybridization, microarrays were washed sequentially.
|
| Scan protocol |
Microarrays were scanned on a G2565CA Microarray Scanner updated to 2 micron resolution (Agilent Technologies, USA). Data were extracted from the resulting TIFF-images using the Feature Extraction software v. 10.7 (Agilent Technologies, USA).
|
| Description |
RNA of PE5-treated OVCAR8 cells (biological replicate 3) was labeled with Cy5, RNA of control OVCAR8 cells (biological replicate 2) was labeled with Cy3, and both RNAs were hybridized to the same microarray.
|
| Data processing |
Raw microarray data were statistically analysed using the software packages Marray, pcaMethods, Limma, and RankProd from Bioconductor (www.bioconductor.org), which uses the R statistical environment and programming language. Specifically, the non-specific signal was removed from the total intensity using the Normexp background correction method with an offset of 20. Then intra-slide normalization was done using the Loess method to make intensities consistent within each array, and inter-slide normalization was performed employing the Aquantiles method to achieve consistency between arrays. After each of these analyses, a quality control analysis of microarray data (RG density plot, MA plot and M boxplot) was performed. Following normalization, the RankProd method was applied to identify differentially expressed genes.
Processed/normalized log2 ratios (test/control) are provided. These values were calculated using both samples of each comparison: samples A and B for the comparison between PE5-treated and control NCI/ADR-RES cells (PE5 vs Control - NCI/ADR-RES), samples C and D for the comparison between onconase-treated and control NCI/ADR-RES cells (Onconase vs Control - NCI/ADR-RES), samples E and F for the comparison between onconase- and PE5-treated NCI/ADR-RES cells (Onconase vs PE5 - NCI/ADR-RES), samples G and H for the comparison between PE5-treated and control OVCAR8 cells (PE5 vs Control - OVCAR8), samples I and J for the comparison between onconase-treated and control OVCAR8 cells (Onconase vs Control - OVCAR8), and samples K and L for the comparison between onconase- and PE5-treated OVCAR8 cells (Onconase vs PE5 - OVCAR8). Therefore, 6 final sets of processed/normalized data are provided, one for each comparison. Only those probes with an FDR adjusted p-value < 0.05 were considered significant.
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| |
|
| Submission date |
Nov 30, 2015 |
| Last update date |
Dec 08, 2016 |
| Contact name |
Maria Vilanova Brugués |
| E-mail(s) |
maria.vilanova@udg.edu
|
| Organization name |
Universitat de Girona
|
| Department |
Biologia
|
| Lab |
Laboratori d'Enginyeria de Proteïnes
|
| Street address |
Maria Aurèlia Capmany 40
|
| City |
Girona |
| ZIP/Postal code |
17071 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE75494 |
Transcriptional profiling of human ovarian cancer cells treated with ribonucleases |
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