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| Status |
Public on Jun 01, 2013 |
| Title |
RA SH-SY5Y_day3 culture_rep2 |
| Sample type |
RNA |
| |
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| Source name |
SH-SY5Y cells_RA differentiation_day3
|
| Organism |
Homo sapiens |
| Characteristics |
culutre day: Day 3
differentiation: RA
treatment: differentiation
|
| Treatment protocol |
As we were interested in the effects of retinoic acid on the growth and differentiation of SH-SY5Y cells, as well as in the toxic effects of MPP+, cultured cells were assigned to three treatment groups: culture in medium only (noRA), culture with medium and 1µM all trans-retinoic acid (RA, Sigma), and culture with medium, 1µM RA and 0.01mM MPP+ (RA/MPP(+), Sigma-Aldrich).
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| Growth protocol |
Human SH-SY5Y neuroblastoma cells were obtained from The European Collection of Cell Culture (ECACC, 94030304, Sigma-Aldrich). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 without L-glutamine (Invitrogen) supplemented with 0.5% fetal calf serum (FCS), 100U/ml penicillin (Sigma) and 0.1mg/ml streptomycin (Sigma) for 8 days at 37°C in 5% CO2. Cells were grown in 96-well plates coated with 0.1mg/ml poly-L-lysine (PLL, Sigma) and 1mg/ml growth factor reduced Matrigel Matrix without phenol red (BD Biosciences). 10,000 cells per well were plated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested for RNA isolation at the time points indicated in Figure 1. Cells were lysed by adding 50µl of Trizol Reagent (Invitrogen) to each well, followed by incubation on ice for 10min. Each time point/treatment condition consisted of 30 wells, divided into three replicates of 10 pooled wells. Phase separation was performed with chloroform and Phase Lock Gel (5 Prime). The final aqueous phase was diluted with an equal volume of 70% ethanol. RNA was isolated using RNeasy Micro columns (Qiagen) according to the manufacturer’s instructions. RNA quantity and purity were determined by NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies) and RNA integrity was determined using the RNA Integrity Number (RIN) measured on an Agilent 2100 Bioanalyzer (Agilent Technologies).
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| Label |
Cy5
|
| Label protocol |
RNA samples were amplified and labeled with either Cy5-CTP or Cy3-CTP using the two-color Low Input Quick Amp Labeling Kit (Agilent Technologies) and purified with the RNA Micro kit according to the manufacturer’s protocol. Quality control was performed where cRNA quantity and dye integration were determined by NanoDrop measurement and cRNA fragment length was investigated with the Bioanalyzer.
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| |
|
| Hybridization protocol |
Labeled cRNA was hybridized on Agilent 4x44K Whole Human Genome arrays (Agilent Technologies, Part Number G4112F) according to the manufacturer’s protocol. Briefly, 825ng of Cy3-CTP or Cy5-CTP labeled cRNA were fragmented for 30min at 60°C in 1X fragmentation buffer (Agilent Technologies) and loaded onto the array in 1X GEx Hybridization Buffer (Agilent Technologies). Arrays were incubated at 60°C in a rotating hybridization chamber for 17h, after which they were washed in 6xSSPE 0.005% N- Lauroylsarcosine (Sigma-Aldrich) for 5min and in 0.06xSSPE 0.005% N-Lauroylsarcosine for one minute. Finally, slides were washed in acetonitril (Sigma-Aldrich) for 30 seconds and dried in a nitrogen flow.
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| Scan protocol |
Microarrays were scanned in an Agilent DNA Microarray Scanner at 5µm resolution at 10% and 100% PMT settings. Scan images were combined and quantified using Agilent Feature Extraction Software (version 9.5.1).
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| Description |
US12302316_251485034455_S01_GE2-v5_95_Feb07_1_1.txt
Gene expression after RA treatment in 3 days of culture
RA.D03.N2
|
| Data processing |
Raw expression data, generated by Feature Extraction software, was imported into the R statistical processing environment using the LIMMA package in Bioconductor (http://www.bioconductor.org). All features for which one or more foreground measurements were flagged as non-uniformity outlier or as saturated outliers were excluded from further analysis. We previously demonstrated that the intensity-based analysis of complex ratio-based designs is more efficient and powerful than the standard ratio-based analysis. We have therefore used an intensity-based analysis for this dataset. The individual signal intensities were extracted from the ratio measurements and 2log transformed intensity measurements were used for further analysis. Normalization between arrays was performed using the quintile algorithm in LIMMA.
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| |
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| Submission date |
Jan 09, 2013 |
| Last update date |
Jun 01, 2013 |
| Contact name |
Joanna Korecka |
| E-mail(s) |
j.korecka@nin.knaw.nl
|
| Organization name |
Netherlands Institute for Neuroscience
|
| Department |
Neuroregeneration
|
| Street address |
Meibergdreef 47
|
| City |
Amsterdam |
| ZIP/Postal code |
1105BA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE43368 |
Phenotypic characterization of retinoic acid differentiated SH-SY5Y cells by transcriptional profiling |
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