|
| Status |
Public on Dec 24, 2016 |
| Title |
DC_mix_D_1 |
| Sample type |
RNA |
| |
|
| Source name |
bone marrow derived dendritic cells, cultured in the presence of rmGM-CSF
|
| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow derived dendretic cells (DCs)
stimulation: stimulated with chimeric peptides in soluble form (mix D)
|
| Treatment protocol |
DCs were stimulated with: (i) chimeric peptides derived from the L. infantum immunogenic proteins CPA, histone H1 and KMP-11 in particulate form (PLGA nanoparticles, mix A), (ii) chimeric peptides and MPLA adjuvant in particulate form (mix B), (iii) chimeric peptides in particulate form surface modified with an octapeptide targeting the TNFRII receptor on DCs surface (mix C), and (iv) chimeric peptides in soluble form (mix D). DCs cultured in medium alone were used as a reference group.
|
| Growth protocol |
DCs were generated from pluripotent bone marrow stem cells obtained from HLA-A2.1 transgenic mice, that were cultured in the presence of rmGM-CSF for 7 days at 37°C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DCs were harvested after 18 h of stimulation. Total RNA was isolated with Trizol Reagent (Invitrogen, Karlruhe, Germany) and purified on a Qiagen RNeasy column (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
Approximately 300 ng of total RNA were used to generate biotinylated complementary RNA (cRNA) for each group using the GeneChip ® WT PLUS Reagent Kit protocol for Whole Transcript (WT) Expression Array, Rev3. Poly-A RNA control added to the RNA test samples as exogenous positive control and the RNA was reverse-transcribed to double stranded cDNA and biotinylated cRNA was synthesized and purified according to the protocol. A 2 nd cycle of cDNA synthesis followed along with a second purification step. Then, 6μg of the single-stranded DNA was fragmented, labeled with the appropriate labeling reagent and hybridized to GeneChip ® Mouse Gene 2.0 ST arrays (Affymetrix, UK).
|
| |
|
| Hybridization protocol |
Hybridization took place for 16 h in an Affymetrix GeneChip ® Hybridization Oven 640. Affymetrix GeneChip® Fluidics Station 450 was then used to wash and stain the arrays with streptavidin-phycoerythrin according to the standard antibody amplification protocol for eukaryotic targets.
|
| Scan protocol |
Arrays were scanned on Affymetrix GeneChip ® Scanner 3000 at 570 nm. Images were acquired using the Affymetrix ® GeneChip ® Command Console ® Software (AGCC).
|
| Data processing |
Standard RMA algorithm steps were followed for background correction, quantile normalization and log summarization of probe sets. Subsequent analysis included differential gene expression analysis using ANOVA (p-value < 0.05). Each comparison was designed in a treated vs. untreated fashion. Probe sets with no annotation or mapping to the same gene were removed from the analysis.
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| |
|
| Submission date |
Dec 23, 2016 |
| Last update date |
Dec 24, 2016 |
| Contact name |
Theresa Busch |
| E-mail(s) |
theresa.Busch@pennmedicine.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Radiation Oncology
|
| Street address |
13400 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE92869 |
Expression data from bone marrow derived DCs stimulated with different peptide-based nanovaccine formulations against L. infantum infection |
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