|
| Status |
Public on Mar 01, 2014 |
| Title |
pDC from donor 3 - CpG stimulated |
| Sample type |
RNA |
| |
|
| Source name |
pDC from donor 3- CpG stimulated
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
|
| Growth protocol |
A. fumigatus conidia (2 x 105) were plated in 48-well plates and grown in pDC medium to hyphae of 10-20 μm average length. pDCs (2 x 105) were then added to the hyphae in a final volume of 300 μl pDC medium for 2 and 4 hr at 37°C. Unstimulated and -CpG-stimulated pDCs were incubated for 2h and 4h, respectively
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). The quantity of total RNA was measured with a spectrophotometer at 260 nanometers, and the RNA integrity was assessed using an RNA 6000 Nano LabChip Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, U.S.A.)
|
| Label |
biotin
|
| Label protocol |
Total RNA (80-500 ng) was reverse transcribed and the single stranded cDNA was amplified using the Ambion WT Expression Kit (Life Technologies, Inc.). The purified cDNA (5.5 μg) was subsequently fragmented and labeled using the GeneChip WT Terminal Labeling Kit (Affymetrix Inc., Santa Clara, CA, U.S.A.)
|
| |
|
| Hybridization protocol |
Labeled cDNA (3.5 ug) was then hybridized to the GeneChip Human Gene 2.0 ST array (Affymetrix, Inc.) using the GeneChip Hybridization Oven 640 (Affymetrix, Inc.) at 60 rotations per minute at 45°C for 16-18 hours. After hybridization, the arrays were washed and stained according to the Affymetrix protocol using a GeneChip Fluidics Station 450 (Affymetrix)
|
| Scan protocol |
The arrays were scanned using the GeneChip Scanner 3000 (Affymetrix).
|
| Data processing |
The data were analyzed using Express Console and Transcriptome Analysis Console (TAC) software (Affymetrix, Inc.). The regulated genes were calculated by dividing the linear intensity value found for each probe from experimental groups (2 or 4 hr of pDC-A. fumigatus interaction and CpG stimulated pDCs) by the linear intensity value found for each probe from the control group (unstimulated pDCs). We considered 1.7 linear fold changes the cut-off to classify the down-regulated and up-regulated genes
|
| |
|
| Submission date |
Feb 28, 2014 |
| Last update date |
Mar 01, 2014 |
| Contact name |
Flavio V Loures |
| E-mail(s) |
loures@icb.usp.br
|
| Organization name |
Universidade de Sao Paulo
|
| Department |
Immunology
|
| Street address |
Rua Deputado joaquim libanio, 216
|
| City |
Sao Paulo |
| State/province |
SP |
| ZIP/Postal code |
04120090 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL16686 |
| Series (1) |
| GSE55467 |
Expression data from pDC infected with Aspergillus fumigatus |
|
