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Status |
Public on May 26, 2011 |
Title |
astrocyte_global mRNA (S803681.H52) |
Sample type |
RNA |
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Source name |
primary human cerebral cortex astrocyte cell line (ScienCell Research Laboratories, cat#1800)
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Organism |
Homo sapiens |
Characteristics |
cell line: primary human cerebral cortex astrocyte cell line (ScienCell Research Laboratories, cat#1800) passage number: between 4 - 6 cell fraction: global cellular fraction
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Growth protocol |
Primary human cerebral cortex astrocyte cells were cultured in cell specific astrocyte medium containing 2% fetal bovine serum, 1% astrocyte growth supplement and 1% penicillin/streptomycin (ScienCell Research Laboratories). Adherent human U-87 MG astrocytoma/glioblastoma cells were cultured under 5% CO2 in DMEM F12 + 1% L-glutamine (2 mM) (Cambrex, Walkersville, MD) supplemented with 10% fetal calf serum (Gibco, Burlington, ON, Canada), 1% penicillin-streptomycin (Gibco), 1% sodium pyruvate (1 mM) (Gibco) and 1% non-essential amino acids (0.1 mM) (Gibco) at 37°C.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA enriched with small RNAs was isolated from the RISC-IP complex and the global cellular fraction using the mirVana kit (Ambion) according to the procedures outlined in the manufacturer’s protocol.
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Label |
biotin
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Label protocol |
Double-stranded cDNA is synthesized from total RNA isolated from cells. Affymetrix’s GeneChip IVT Express kit was used for cDNA synthesis and in vitro transcription to produce biotin-labeled cRNA from the cDNA. The cRNA is fragmented before hybridization. As outlined in detail in http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf
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Hybridization protocol |
A hybridization cocktail is prepared, including the fragmented target, and probe array controls. It is then hybridized to the probe array during a 16-hour incubation. As outlined in detail in http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf
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Scan protocol |
Once the probe array has been hybridized, washed, and stained, it is scanned. Each workstation running Affymetrix Microarray Suite or GCOS can control one scanner. The software defines the probe cells and computes an intensity for each cell. Each complete probe array image is stored in a separate data file identified by the experiment name and is saved with a data image file (.dat) extension.
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Description |
The concentration of RNA samples was determined using Nanodrop ND-1000 Spectrophotometer and the RNA quality of 2 samples was checked on RNA Nano chip using Agilent 2100 Bioanalyzer. All RNA samples displayed no degradation with two sharp ribosomal peaks. Affymetrix’s GeneChip IVT Express kit was used for cDNA synthesis and in vitro transcription.
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Data processing |
The data were processed by Affymetrix Software Command Console and Expression Console using RMA algorithms which consisted of three steps: background correction, quantile normalization (each performed at the individual probe level), and robust linear model fit using log-transformed intensities (at the probe set level). The RMA normalized expression values in the Matrix are the average of 4 replicates for each of the 4 samples. The overall selection criteria for presence used genes with expression values larger than a threshold 3.5 in more than 50% of samples (2 out of 4).
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Submission date |
Apr 26, 2010 |
Last update date |
May 26, 2011 |
Contact name |
Joanna Moser |
Organization name |
University of Calgary
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Department |
Biochemistry and Molecular Biology
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Lab |
Dr. Marvin Fritzler
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Street address |
3330 Hospital Drive N.W.
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City |
Calgary |
State/province |
AB |
ZIP/Postal code |
T2N 4N1 |
Country |
Canada |
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Platform ID |
GPL570 |
Series (1) |
GSE21514 |
The mRNA and microRNA expression profile of the RNA-induced silencing complex in human U-87 astrocytoma cells and primary human astrocytes |
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