|
| Status |
Public on Dec 18, 2007 |
| Title |
BOEC, normal 15 |
| Sample type |
RNA |
| |
|
| Source name |
BOEC, normal 15
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: blood outgrowth endothelial cell
status: normal
race: African American
age: 27
sex: M
|
| Treatment protocol |
BOEC were always harvested at passage 3, which comprised a nominal million-fold expansion since establishing culture and amounted to ~3x107 BOEC. They were always harvested 4 hours after the last change of culture medium, and always when they were at 85-90% confluent.
|
| Growth protocol |
We obtained citrated fresh peripheral blood (50-100 ml) from each donor. For donors outside the Minneapolis metropolitan area, blood was shipped by same-day express delivery in Saf-T-Pak cartons that had been pre-equilibrated to room temperature. Samples were processed immediately upon arrival at the University of Minnesota. Blood buffy coat mononuclear cells were prepared and cultured on rat collagen I, in presence of endothelial cell growth factors, as previous described in detail 15,16. All samples were handled identically
|
| Extracted molecule |
total RNA |
| Extraction protocol |
We prepared BOEC lysates by adding 10 ml trizol (Gibco) to each T75 flask to solubilize the sample. As necessary, trizol lysates were stored in liquid nitrogen. We then isolated total RNA, and cleaned it using an RNeasy kit (Qiagen, CA).
|
| Label |
biotin
|
| Label protocol |
We used the Invitrogen SuperScript Choice system to reverse transcribe and synthesize double-stranded cDNA. For in vitro transcription and biotin-labeling, we synthesized biotin-labeled cRNA using the Enzo Life Sciences BioArray High Yield RNA Transcript Labeling kit. Then fragmentation of cRNA was done by standard protocol, and fragmentation was verified by gel electrophoresis.
|
| |
|
| Hybridization protocol |
After we prepared biotin-labeled cRNA fragments, samples were turned over immediately to our Microarray Core Facility which used Gene Chip Hybridization Oven 640, Affymetrix FS4000 Fluidics Station for staining with a streptavidin-fluorochrome probe and washing,
|
| Scan protocol |
An Affymetrix High Resolution Scanner was used according to the manufacturer's standard manual
|
| Description |
BOEC (blood outgrowth endothelial cells) from individuals that do not have sickle cell anemia
|
| Data processing |
For raw microarray data, we used the RMA (robust multi-array average) method to summarize expression values from probe pair values; data were background-adjusted, quantile normalized (global scaling across multiple arrays). Expression measures were summarized based on log transformed PM (perfect match) values using Median Polish algorithm. Then, we used locally weighted scatterplot smoothing (LOWESS) to do within-array normalization. RMA and LOWESS were performed in Genedata Expressionist Pro3.1® (Basel, Switzerland).
|
| |
|
| Submission date |
Dec 13, 2007 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Robert P. Hebbel |
| E-mail(s) |
enens001@umn.edu
|
| Phone |
612-6244620
|
| Fax |
612-625-8105
|
| Organization name |
University of Minnesota
|
| Department |
Medicine
|
| Lab |
Vascular Biology Center
|
| Street address |
MMC 480, 420 Delaware St. SE
|
| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
| |
|
| Platform ID |
GPL96 |
| Series (2) |
| GSE9877 |
Genetic Endothelial Systems Biology of Sickle Stroke Risk |
| GSE17078 |
Cell Adhesion Molecule 1 (CADM1): A Novel Risk Factor for Venous Thrombosis |
|
| Relations |
| Reanalyzed by |
GSE86363 |
