|
| Status |
Public on Nov 19, 2009 |
| Title |
SH-SY5Ycells_bilirubin_treated_140nM_4hours_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Human neuroblastoma SH-SY5Y cells, treated with free bilirubin (Bf) 140nM, for 4 hours
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-SY5Y
atcc: CRL-2266
cell type: neuroblastoma
treatment: free bilirubin
treatment time: 4 hours
|
| Treatment protocol |
The growth medium was replaced with experimental medium, which was either 140 nM Bf medium (Bf 140 nM samples) or 0.6 % DMSO medium (DMSO 0.6 % samples), and cells were allowed to grow. After 4 h, cells were collected and processed for microarray analysis. Separate flasks of cells were used for each of the treatments and assays. Experiments were performed using at least three independent biological replicates.
|
| Growth protocol |
SH-SY5Y human neuroblastoma cells (ATCC CRL-2266) were cultured in a humidified incubator at 37°C and 5 % CO2 in F12-EMEM (1:1) containing 15 % foetal bovine serum (FBS), 2 mM L-glutamine, 25 µg/mL gentamicin, 100 U/ml penicillin, and 100 µg/mL streptomycin at 37°C with 5 % CO2. Cells were seeded at 6 x 10e6 cells in 75 cm2 flasks and grown for 24 h (cells reached 70-80% confluence).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the TRIzol reagent (Invitrogen) following the manufacturer’s instructions. It was then purified using the RNeasy mini kit (Qiagen). The quality of total RNA was assessed using a bioanalyzer (Agilent 2100; Agilent Technologies) and RNA was quantified by using a ND-1000 Nanodrop spectrophotometer.
|
| Label |
Biotin
|
| Label protocol |
10 μg of each total RNA sample was labelled according to the standard one-cycle amplification and labelling protocol developed by Affymetrix (Santa Clara, CA).
|
| |
|
| Hybridization protocol |
Labelled cRNA was hybridized on Affymetrix GeneChip Human U133A 2.0 Arrays containing over 14,500 transcripts. Hybridized GeneChips were stained and washed using the GeneChip Fluidic Station 450.
|
| Scan protocol |
GeneChips were scanned with the GeneChip Scanner 3000 7G. Cell intensity values and probe detection calls were computed from the raw array data using the Affymetrix GeneChip Operating Software (GCOS).
|
| Description |
Bf140nM4h_6
|
| Data processing |
Further data processing was performed in the R computing environment (http://www.r-project.org/, version R 2.5.0 for Windows) using packages from the BioConductor software project (http://www.bioconductor.org/). Variance-stabilizing normalization was applied, using the justvsn function from the vsn library. Normalized data were then filtered based on the Affymetrix detection call, so that only probes that had a Present call in at least one of the arrays were retained. Data were then imported in the MultiExperiment Viewer (MeV) software (version 4.0.01 for Windows XP), and statistical analysis was performed with the SAM (Significance Analysis of Microarrays) module.
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| |
|
| Submission date |
Jun 23, 2009 |
| Last update date |
Nov 19, 2009 |
| Contact name |
Paola Roncaglia |
| Organization name |
SISSA/ISAS (International School for Advanced Studies)
|
| Department |
Neurobiology
|
| Street address |
via Bonomea 265
|
| City |
Trieste |
| State/province |
TS |
| ZIP/Postal code |
34136 |
| Country |
Italy |
| |
|
| Platform ID |
GPL571 |
| Series (2) |
| GSE16767 |
Transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells: 4h |
| GSE16768 |
Transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells |
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