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Status |
Public on Dec 01, 2008 |
Title |
Day7_01173; VaxDesign Mimic experiment |
Sample type |
RNA |
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Source name |
Frozen stocks of autologous PBMCs were used as a source of lymphocytes.
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Organism |
Homo sapiens |
Characteristics |
Yellow Fever unexposed volunteer
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Biomaterial provider |
Donald R. Drake,VaxDesign Corporation, 12612 Challenger Parkway, Suite 365, Orlando, FL 32826, USA
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Treatment protocol |
Purified CD4+ T cells were co-cultured in the Mimic System with untreatedDCs at a ratio of 60:1 for 7 days
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was purified using RNA extraction kits from Qiagen. Quantification was performed using NanoDrop Technologies spectrophotometer and RNA quality was assessed using the Experion automated electrophoresis system from Bio-Rad.
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Label |
biotin
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Label protocol |
Total RNA as amplified and labeled using the Illumina TotalPrep RNA Amplication kit which is based on the Eberwine amplification protocol. It involves a first cDNA synthesis step followed by in vitro transcription of cRNA.
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Hybridization protocol |
The biotinylated cRNA was hybridized onto Illumina Human RefSeq-8 BeadChips v2 at 58oC for 20 hours.
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Scan protocol |
Fluorescent array images were collected with Illumina BeadStation 500GX scanner fluorescent scanner and image intensity data were extracted using Illumina BeadStudio version 3 software.
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Description |
PBMCs used in the assays were acquired from normal healthy donors enrolled in a VaxDesign Corporation apheresis study program (protocol CRRI 0906009). Blood collections were performed at Florida’s Blood Centers (Olrando, FL) using standard techniques. The enriched leukocytes were isolated and cryopreserved. Human DCs were prepared as described in Gaucher et al. “Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses , J. Exp. Med XXX. After 5 days of culture with IL-4 and GM-CSF, the DCs were infected with a 1:100 dilution of the live-attenuated YF-VAX® vaccine. After 24 hrs, 5% human AB serum was added to the media to quench the infection. Alternatively, some of the DCs we not infected. On the sixth day, all DCs were matured with an overnight treatment of 25 ng/mL TNF- (R&D Systems).
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Data processing |
Illuma probe data were exported from BeadStudio as raw data and were screened for quality. Samples failing chip visual inspection and control examination were removed. The R software was used to quantile normalize the probe intensities, and to minimum-replace (surrogate-replacement policy) values below background using the mean background vale of the probe controls rounded up to the rearest power of 2, determined from the microarray quality controls to reduce noise and reduce "over-inflated" expression ratios in subsequent steps.
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Submission date |
Nov 19, 2008 |
Last update date |
Nov 27, 2008 |
Contact name |
Bastian Robert Angermann |
E-mail(s) |
angerb@gmx.de
|
Fax |
5143437854
|
Organization name |
Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CR-CHUM) Saint-Luc
|
Lab |
Laboratoire d'immunologie
|
Street address |
264 René-Lévesque Est
|
City |
Montréal |
State/province |
Quebec |
ZIP/Postal code |
H2X 1P1 |
Country |
Canada |
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|
Platform ID |
GPL6883 |
Series (1) |
GSE13699 |
Immune response to the yellow fever vaccine 17D. |
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