|
| Status |
Public on Sep 17, 2014 |
| Title |
A549-2 weeks-2-rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
RNA from A549 cells exposed to 2 µM of sodium arsenite for 2 weeks replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: A549 human epithelial lung carcinoma cells
treatment: 2 µM of sodium arsenite
time: 2 weeks
|
| Treatment protocol |
Cells were exposed to sodium arsenite (NaAsO2, Sigma) at concentrations of 0.08 µM, 0.4 µM and 2 µM for 1, 2 and 8 weeks. Exposure to sodium arsenite started 6 hours after the cells were seeded into the culture flasks to ensure that sodium arsenite did not affect attachment. The final concentrations of sodium arsenite were obtained by the appropriate dilution with media. Medium was changed after three days thereby providing a new dose of sodium arsenite to the cells. All experiments were performed in duplicate.
|
| Growth protocol |
A549 cells (human epithelial lung carcinoma cells; obtained from the American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and 1% penicillin/streptomycin (Sigma) and maintained at 37 °C in a 5% CO2 atmosphere. Cells were seeded at a concentration of 4000 cells/cm2 in T25 flasks and trypsinized weekly.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After removal of the medium, cells were lysed with TRIzol (Invitrogen) and total RNA was extracted using 0.5 ml TRIzol according to the manufacturer’s instructions. RNeasy Mini Kits (Qiagen, Westburg bv, The Netherlands) were used to purify total RNA from salts and residual DNA. From each sample, RNA quantity was measured spectrophotometrically using the NanoDrop 1000, and integrity was determined using the Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). All samples had on OD 260/280 ratio between 1.8 and 2.0, and held an RNA integrity number > 8.
|
| Label |
Biotin
|
| Label protocol |
cDNA was prepared using the Affymetrix One-Cycle cDNA synthesis kit (Affymetrix, Santa Clara). cDNA synthesis and labeling was performed according to the manufacturer’s rocedures. Subsequent labeling of the samples was conducted by synthesis of Biotin-labeled complementary RNA (cRNA) using the GeneChip IVT labeling kit (Affymetrix). Purified cRNA was quantified using a spectrophotometer, and unfragmented samples were checked on the Bioanalyzer. Subsequently, cRNA samples were fragmented for target preparation according to the Affymetrix manual and checked on the Bioanalyzer.Samples were stored at 20 C until ready to perform hybridization. cDNA synthesis kit (Affymetrix, Santa Clara). cDNA synthesis and labeling was performed according to the manufacturer’s procedures. Subsequent labeling of the samples was conducted by synthesis of Biotin-labeled complementary RNA (cRNA) using the GeneChip IVT labeling kit (Affymetrix). Purified cRNA was quantified using a spectrophotometer, and unfragmented samples were checked on the Bioanalyzer. Subsequently, cRNA samples were fragmented for target preparation according to the Affymetrix manual and checked on the Bioanalyzer. Samples were stored at 20 C until ready to perform hybridization.
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| |
|
| Hybridization protocol |
cRNA targets were hybridized on high-density oligonucleotide gene chips (Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays) according to the Affymetrix Eukaryotic Target hybridization manual. The gene chips were washed and stained using the Affymetrix Fluidics Station 450/250 and Genechip Operating Software
|
| Scan protocol |
Chips were scanned by means of an Affymetrix GeneArray scanner
|
| Description |
Sample name: 2-2.0-R-2a
|
| Data processing |
The Affymetrix CEL files were imported into R v2.15.0 [24] using the “affy” library within BioConductor (v2.9). Probesets were reannotated to EntrezGene genes using the BrainArray custom CDF v15.0 annotations. The quality of the arrays was assessed through box plots, fitPLM, NUSE, RLE, clustering/heat maps, PCA and correlation plots. No technically deviating arrays were detected. Probeset intensities were normalized using the RMA algorithm. For detecting differentially expressed genes, the “limma” library was used to construct a linear model containing coefficients for all factor combinations. The resulting p-values were FWER-corrected using the False Discovery Rate (FDR) approach.
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| |
|
| Submission date |
Feb 08, 2013 |
| Last update date |
Sep 17, 2014 |
| Contact name |
Simone G van Breda |
| E-mail(s) |
s.vanbreda@maastrichtuniversity.nl
|
| Phone |
0031433882127
|
| Organization name |
Maastricht University
|
| Department |
Toxicogenomics
|
| Street address |
Universiteitssingel 50
|
| City |
Maastricht |
| State/province |
Limburg |
| ZIP/Postal code |
6229 ER |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16356 |
| Series (2) |
| GSE44169 |
Sodium arsenite induced gene expression changes in A549 human epithelial lung carcinoma cells |
| GSE44174 |
Sodium arsenite induced changes in A549 human epithelial lung carcinoma cells |
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