|
| Status |
Public on Jun 01, 2009 |
| Title |
single-cultured pMBMEC - 1 |
| Sample type |
RNA |
| |
|
| Source name |
single-cultured mouse brain endothelial cells
|
| Organism |
Mus musculus |
| Characteristics |
for each preparation cortices from ten 4 week old female C57BL/6 mice (Charles River, L’Arbresle, France)
|
| Treatment protocol |
as above
|
| Growth protocol |
mouse brain microvascular endothelial cells were isolated with or without culture as described above
|
| 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
|
| Label |
biotin
|
| 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.
|
| |
|
| 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
|
| 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.
|
| Description |
gene expression from 5 day cultures on transwell filters of mouse brain microvascular endothelial cells
|
| 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
|
| Phone |
+41 44 635 50 44
|
| Fax |
+41 44 635 68 14
|
| Organization name |
University of Zurich
|
| Department |
Institute of Physiology
|
| Street address |
Winterthurerstr. 190
|
| City |
Zurich |
| ZIP/Postal code |
CH-8057 |
| Country |
Switzerland |
| |
|
| 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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