|
| Status |
Public on May 20, 2010 |
| Title |
brain_endothelial_cells_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
endothelial cells from dorsolateral prefrontal cortex
|
| Organism |
Homo sapiens |
| Characteristics |
sex: m; age (years): 44
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Endothelial cells were collected from seven humans and neuronal cells from six humans which partially overlap. Fifteen µm thick cryosections (Bright Technologies) of dorsolateral prefrontal cortex were cut, mounted onto microscope slides, air dried, and acetone fixed. The sections were stained either with a polyclonal antibody to detect neurons (Neurofilament 160/200kDa mouse; Cambridge Bioscience 13-1300) or endothelial cells (von Willebrand factor (VWF) rabbit; Chemicon AB7356) in conjunction with a secondary fluorescently labeled (Cy2 goat-anti-mouse (Jackson Immunoresearch) for the neuron and Cy3 goat anti-rabbit (Jackson Immunoresearch) for the endothelial staining) polyclonal antibody in the presence of RNAse inhibitor (Amersham Biosciences). The stained sections were subsequently washed with phosphate buffered saline (PBS, Ambion) and dehydrated by incubation in increasing ethanol concentrations (30%, 50%, 70%, 90%, and absolute ethanol). Approximately 1,000 neurons or an equivalent tissue area of endothelium per individual were microdissected using a laser capture microdissection microscope (P.A.L.M.), RNA was extracted using a commercially available RNA extraction kit (P.A.L.M.).
|
| Label |
biotin
|
| Label protocol |
Amplification of mRNA was carried out in three consecutive linear amplifications using the RiboAmpHS kit (Arcturus). During the last amplification, biotin-labeled UTP was incorporated. The quality of the amplified RNA was assessed with a Bioanalyser Nanochip (Agilent).
|
| |
|
| Hybridization protocol |
Hybridisation (16 hours), washing and staining were done by following the instructions of the chip manufacturer.
|
| Scan protocol |
The chips were scanned with a GeneChip scanner system. Microarray image data was analyzed with the GeneChip® Operating Software
|
| Description |
individual suffered sudden death, without brain injury
|
| Data processing |
Gene probe set expression values were calculated using the Robust Multichip Average for small RNA quantities (srma) algorithm. As probe weights for signal summary statistic calculation we used the inverse of the probe specific coefficient of variation calculated across chips. Probe sets were defined as expressed if the P-value determined by a signed rank test of perfect match versus mismatch signal distributions was lower or equal 0.065 in at least three chip experiments in a given cell type. Probe sets that showed statistically significant higher expression in neurons or endothelial cells in two-sided t-tests were considered as preferentially neuronal or endothelial expressed genes, respectively.
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| |
|
| Submission date |
Jul 30, 2008 |
| Last update date |
May 06, 2010 |
| Contact name |
Thomas Giger |
| E-mail(s) |
giger@eva.mpg.de
|
| Organization name |
Max-Planck-Institute of Evolutionary Anthropology
|
| Department |
Evolutionary Genetics
|
| Street address |
Deutscher Platz 6
|
| City |
Leipzig |
| ZIP/Postal code |
04106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE12293 |
Evolution of neuronal and endothelial transcriptomes in primates |
|
| Relations |
| Reanalyzed by |
GSM318416 |
