|
| Status |
Public on Dec 18, 2008 |
| Title |
endothelial_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
human endothelial cells isolated from postmortem dorsolateral prefrontal cortex
|
| Organism |
Homo sapiens |
| Characteristics |
Cell type: endothelial, age:32, gender:male, diagnostic group: control, PMI:13h
|
| Biomaterial provider |
Stanley Medical Research Institute
|
| Treatment protocol |
15um tissue sections were cut onto polyethylene naphthalate membrane slides (Zeiss) and fixed for 10 minutes in acetone. After air drying, sections were incubated with either rabbit anti-von Willebrand factor (Chemicon) or mouse anti-neurofilament-160/200kD for 5 minutes, followed by brief washing in RNase-free phosphate buffered saline (PBS) and incubation in secondary antibody for 5 minutes (Cy3-conjugated goat anti-rabbit IgG or Cy2-conjugated goat anti-mouse IgG (Jackson Immunoresearch). All antibodies were used at 1:20 dilution with 1unit/ml RNase inhibitor (GE Healthcare) in RNase-free PBS (Ambion). These incubation conditions were found to give the best staining for cell identification together with optimal RNA preservation. Following antibody incubation and brief washing in PBS, sections were then dehydrated through ethanol series and cell capture was initiated immediately. Laser capture microdissection was carried out using the PALM microlaser system [22]. 1000 neurons were captured from each subject, in two batches of 500, or an equivalent area of vascular endothelium (approximately 40 0000 m2). Pyramidal neurons were selected based on staining and morphology.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using PALM extraction kit (Zeiss) according to instructions
|
| Label |
biotin
|
| Label protocol |
RNA was amplified through 2 rounds using the RiboAmp HS kit (Arcturus) according to instructions. Amplified RNA was converted to cDNA using Round 2 components of the RiboAmp HS kit, labelled by in vitro transcription in the presence of biotinylated UTP (Codelink Expression Assay kit, GE Healthcare), and purified using YM-30 columns (Microcon).
|
| |
|
| 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 |
amplified RNA
|
| Data processing |
Expression measures were computed for each of the probesets on each of the GeneChips in the dataset using the robust multichip average (RMA) method, which is implemented in the BioConductor package ‘Affy’. RNA digestion plots were also generated using Affy package. A linear regression of expression values on the logarithm (base 2) of slope of the RNA digestion plots for each probeset was performed and the residuals from the regression were assigned as expression values for further analysis.
|
| |
|
| Submission date |
Sep 05, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Laura Wiseman Harris |
| Organization name |
University of Cambridge
|
| Department |
Insitute of Chemical Engineering and Biotechnology
|
| Street address |
Tennis Court Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB22 4RA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE12679 |
Laser capture microdissection of endothelial and neuronal cells from human dorsolateral prefrontal cortex |
|
| Relations |
| Reanalysis of |
GSM309042 |
| Reanalyzed by |
GSE119087 |
