10 protocols
AccessionType
array scanning and feature extraction protocol
Title: Affymetrix CEL analysis. Description:
specified_biomaterial_action
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.
bioassay_data_transformation
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.
bioassay_data_transformation
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. A False Discovery Rate (FDR) of about 10% (i.e. 9.317%) was applied to detect significantly differentially expressed genes.
labeling
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).
grow
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).
nucleic_acid_extraction
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.
specified_biomaterial_action
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 1 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.
grow
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 106 cells in 75 cm2 flasks and grown for 24 h (cells reached 70-80% confluence).
specified_biomaterial_action
The medium was then 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 24 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 at least in three independent biological replicates.