|
| Status |
Public on Nov 23, 2008 |
| Title |
Subject ID 138 time 12hr no stimulation |
| Sample type |
RNA |
| |
|
| Source name |
138_12hr_media
|
| Organism |
Homo sapiens |
| Characteristics |
PBMC
Time: 12
Stimulation: none
|
| Growth protocol |
PBMCs from 2 healthy unvaccinated donors were isolated and plated at 1 x 106 cells per well in 48-well plates with 1 ml RPMI with 10% FBS and penicillin/streptomycin. The cells were cultured in the presence or absence of YF-17D at a MOI of 1. After 3 and 12 hours, RNA was isolated from the cells and processed for microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following culture, RNA was isolated with 1 ml of TRIzol following the manufacturer's protocol (Cat# 15596-026; Invitrogen Life Technologies).
|
| Label |
Biotin
|
| Label protocol |
Total RNA sample quality was evaluated by spectrophotometer to determine quantity, protein contamination and organic solvent contamination, and an Agilent 2100 Bioanalyzer was used to check for RNA degradation. Two-round IVT amplification and labeling was performed starting with 50 ng intact, uncontaminated total RNA per sample, following the Affymetrix protocol (http://www.affymetrix.com/Auth/support/downloads/manuals/expression_print_manual.zip) (Affymetrix, CA).
|
| |
|
| Hybridization protocol |
After hybridization on Human Genome U133 A 2.0 Arrays for 16 hours at 45 C and 60 rpm in a Hybridization Oven 640, slides were washed and stained with a Fluidics Station 450.
|
| Scan protocol |
Scanning was performed on a 7th generation GeneChip Scanner 3000, and the Affymetrix GCOS software was used to perform image analysis and generate raw intensity data.
|
| Description |
Gene expression from YF-17D Vaccinee
|
| Data processing |
For this analysis, we used Affymetrix Human Genome U133 A 2.0 Array, but instead of using Affymetrix’s sequence clusters to define genes, gene sequence clusters were RMA pre-processed and normalized with the UniGene (Build 199, January 16, 2007) Custom CDF Version 9 available from http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/CDF_download.asp to yield a list of 11,639 genes.
|
| |
|
| Submission date |
Nov 05, 2008 |
| Last update date |
Nov 20, 2008 |
| Contact name |
Troy D Querec |
| Organization name |
Emory University
|
| Department |
Emory Vaccine Rearch Center
|
| Lab |
Pulendran
|
| Street address |
954 Gatewood Rd
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30329 |
| Country |
USA |
| |
|
| Platform ID |
GPL7566 |
| Series (2) |
| GSE13484 |
Profiling 11,639 genes in PBMCs from 2 donors: U133 A 2.0 Custom CDF Version 9 |
| GSE13486 |
Time Course of Individuals Vaccinated with YF-17D |
|
