|
| Status |
Public on Dec 21, 2010 |
| Title |
pDCs-NYVAC-C-KC-rep9 |
| Sample type |
RNA |
| |
|
| Source name |
Plasmacytoid Dendritic Cells (pDCs)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Plasmacytoid Dendritic Cells (pDCs)
viral infection: NYVAC-C-KC
|
| Treatment protocol |
cDCs and pDCs were infected for 1 hour and after washing left in medium for 6 hours before RNA extraction.
|
| Growth protocol |
cDCs and pDCs were obtained by MACS cell sorting from freshly isolated peripheral blood mononuclear cells (PBMC). Cells were used directly after isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA quantification was performed using a spectrophotometer (NanoDrop Technologies) and RNA quality was assessed using the Experion automated electrophoresis system (Bio-Rad).
|
| Label |
biotin
|
| Label protocol |
Total RNA was amplified and labeled using the Illumina TotalPrep RNA Amplification kit, which is based on the Eberwine amplification protocol.
|
| |
|
| Hybridization protocol |
The biotinylated cRNA was hybridized onto Illumina Human RefSeq-8 BeadChips V2 and V3 at 58oC for 20 hrs
|
| Scan protocol |
Fluoresent array images were collected using Illumina BeadStation 500GX scanner and image intensity data were extracted using Illumina BeadStudio v3.
|
| Description |
pDCs infected by restricted poxvirus NYVAC containing HIV clade C gag, pol, nef and env genes whose replication capacity was restored by inserting two viral host range genes, K1L and C7L
|
| Data processing |
Illumina probe data were exported from BeadStudio as raw data and were screened for quality. Samples failing chip visual inspection and control examination were removed. Probeset from the two Illumina platforms were mapped to a common probeset Id using a mapping file provided by Illumina. One dataset containing probeset common to both platforms was then used for pre-processing and subsequent analysis steps. The R software was used to quantile-normalized the probe intensities, and to minimun replace (surrogate-replacement policy) values below backround using the mean backround value of the built-in Illumina probe controls as an alternative to background substraction (which may introduce negative values) to reduce ‘over inflated’ expression ratios in subsequent steps.
|
| |
|
| Submission date |
Dec 21, 2010 |
| Last update date |
Dec 21, 2010 |
| Contact name |
Ali Filali |
| E-mail(s) |
afilali@vgtifl.org
|
| Organization name |
Vaccine & Gene Therapy Institute of Florida
|
| Street address |
11350 SW Village Parkway, 3rd Floor
|
| City |
port st-lucie |
| State/province |
Florida |
| ZIP/Postal code |
34987 |
| Country |
USA |
| |
|
| Platform ID |
GPL6883 |
| Series (1) |
| GSE26239 |
Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors |
|
