|
| Status |
Public on Jul 01, 2007 |
| Title |
HUVEC control_sample K |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC monolayer
|
| Organism |
Homo sapiens |
| Characteristics |
human umbilical vein endothelial cells, passages 3
|
| Biomaterial provider |
Clonetics
|
| Treatment protocol |
HUVEC cells cultured in a 1:2 mixture of endothelial growth medium (EGM (Clonetics) and M199 medium (PAA, Cölbe, Germany) supplemented with 6.7% fetal bovine serum, gentamicin, amphotericin and Liquemin® N5000 (Roche, Grenzach-Wyhlen, Germany).
|
| Growth protocol |
Twenty-four hours prior to addition of C. albicans, HUVEC cells were washed and placed in antibiotic-free medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (5 µg/experiment) from medium-stimulated controls or HUVEC exposed to C. albicans out of 4 independent experiments was prepared using RNeasy kits from Qiagen (Hilden, Germany) according to the manufacturer’s instructions. cDNA was prepared from 5 µg total RNA using the SuperScript Choice System kit (Invitrogen, Karlsruhe, Germany) with a T7-(dT)24 primer for first-strand synthesis.
|
| Label |
biotin
|
| Label protocol |
cRNA was synthesized from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY).20µg of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer (40 mmol/L Tris-acetate, pH 8.1, 100 mmol/l Potassium acetate, 30 mmol/L Magnesium acetate).
|
| |
|
| Hybridization protocol |
10 µg of fragmented cRNA were together with control cRNAs and grid alignment oligonucleotides hybridized under constant rotation at 45°C over night (16h) to oligonucleotide. Arrays were washed and stained (label: SAPE; Gene Chip Washing and Staining Hybridization kit, Affymetrix) using the Gene Chip Fluidics Station 450 (Affymetrix).
|
| Scan protocol |
Fluorescent signals were detected by the Affymetrix GeneChip Scanner 3000. Microarrays of this series were all scaled to a TGT value of 100.
|
| Description |
untreated HUVEC control
|
| Data processing |
Microarray data were analyzed using MicroArray Suite (MAS) software (Affymetrix). First, background-adjusted raw intensities accounting for non-specific binding by removing probe sets with insignificant differences between single perfect-matching (PM) and mismatching (MM) probes were created. Single raw values were calculated for each probe set from the median of PM/MM discrimination values. Fold-changes (log ratio changes) and "change p values" based on a signed rank test were determined for each experiment. Only genes with a change p≤0.05 for up-regulated or a change p≥0.95 for down-regulated genes in at least 3 of 4 experiments and mean log ratio changes (calculated as mean of log ratio changes of all experiments with significant change p values) of at least 2.5 or -2.5 were considered.
|
| |
|
| Submission date |
Mar 23, 2007 |
| Last update date |
Mar 28, 2007 |
| Contact name |
Dorothee Viemann |
| E-mail(s) |
Viemann.Dorothee@mh-hannover.de
|
| Phone |
+49-511-532 - 7823
|
| Organization name |
Hannover Medical School
|
| Department |
Department of Pediatric Pulmonology, Allergy and Neonatology
|
| Street address |
Carl-Neuberg-Straße 1
|
| City |
Hannover |
| State/province |
Deutschland |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE7355 |
Candida-induced expression profile in HUVEC |
|
