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Status |
Public on Jun 01, 2009 |
Title |
co-cultured pMBMEC - 3 |
Sample type |
RNA |
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Source name |
co-cultured mouse brain endothelial cells
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Organism |
Mus musculus |
Characteristics |
for each preparation cortices from ten 4 week old female C57BL/6 mice (Charles River, L’Arbresle, France)
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Treatment protocol |
as above
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Growth protocol |
mouse brain microvascular endothelial cells were isolated with or without culture as described above
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted using RNAready kit (BioDiagnostik, University Bern, Bern, Switzerland) according to the manufacturer protocol including an on-column DNase digestion
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Label |
biotin
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Label protocol |
cDNAs were prepared by reverse transcription (RT) (WT-Ovation Pico System, NuGEN) according to the manufacturer’s protocol from total isolated RNAs. Fragmented and biotin labelled single-stranded cDNA targets were generated with the FL-Ovation cDNA Biotin Module V2 (NuGEN, 4200-12) according to the manufacturer’s protocol.
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Hybridization protocol |
The biotin-labelled cDNA targets were mixed in 220 µL of Hybridization Mix (Affymetrix Inc., P/N 9007200) containing Hybridization controls and Control Oligonucleotide B2 (Affymetrix Inc., P/N 900454). Samples were hybridized to GeneChip® arrays for 18 h at 45 ºC
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Scan protocol |
An Affymetrix GeneChip® Scanner 3000 (Affymetrix Inc.) was used to measure fluorescent intensities emitted by labelled targets and signals were saved to a CEL file format.
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Description |
gene expression from 5 day cultures on transwell filters of mouse brain microvascular endothelial cells co-cultured with 3 week mouse brain glial cultures in the lower well in non-contact
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Data processing |
CEL file date were normalized using RMAExpress 0.5 (Robust Multichip Average) (Bolstad et al 2003) and Custom CDF version 10 (Mm430_Mm_REFSEQ.cdf) (Dai et al 2005). The RMAExpress options background-adjustment and quantile normalization were enabled. To exclude probe sets in the lower end of the signal range which have large signal variation, a cutoff value was defined as the maximal signal value obtained from non-murine Affymetrix-control probe sets multiplied by a factor of 1.2. In this data set, the cutoff value was log25.78. Probe sets with a signal below cutoff in every array of the corresponding comparison, as well as Affymetrix control probe sets (prefix of “AFFX”) were excluded from further analysis.
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Submission date |
Jan 12, 2009 |
Last update date |
Jan 12, 2009 |
Contact name |
Francois Verrey |
E-mail(s) |
verrey@access.uzh.ch
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Phone |
+41 44 635 50 44
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Fax |
+41 44 635 68 14
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Organization name |
University of Zurich
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Department |
Institute of Physiology
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Street address |
Winterthurerstr. 190
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City |
Zurich |
ZIP/Postal code |
CH-8057 |
Country |
Switzerland |
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Platform ID |
GPL8059 |
Series (1) |
GSE14375 |
Culture induced changes in Blood-Brain Barrier transcriptome: implications for amino acid transporters in vivo |
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