|
| Status |
Public on Dec 08, 2016 |
| Title |
Control (Cy5) Onconase (Cy3) - NCI/ADR-RES |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control NCI/ADR-RES cells. Biological replicate 4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: NCI/ADR-RES
cell type: MDR 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 |
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 |
Onconase-treated NCI/ADR-RES cells. Biological replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: NCI/ADR-RES
cell type: MDR ovarian cancer cells
treatment: Onconase
|
| 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 control NCI/ADR-RES cells (biological replicate 4) was labeled with Cy5, RNA of Onconase-treated NCI/ADR-RES cells (biological replicate 1) 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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