|
| Status |
Public on Dec 05, 2012 |
| Title |
m_DCs_PolyIC1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Splenic dendritic cell, polyI:C-stimulated, 8 hours
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
cell type: splenic dendritic cell, Flt3L expanded in vivo
treatment: In vitro polyI:C stimulation, 8 hours
|
| Treatment protocol |
Cells were infected with H1N1 influenza A/Puerto Rico/8/34 at an MOI=10, stimulated with 30 ug/mL polyI:C, or cultured in media alone for 8 h (RPMI-1640, 10% heat-inactivated FBS, 2 mM L-glutamine, penicillin, streptomycin, 100 μM β-mercaptoethanol).
|
| Growth protocol |
C57Bl/6 mice were injected subcutaneously between the shoulder blades with 6x106 B6-melanoma cells expressing Flt3L to expand the dendritic cell compartment. After 2 weeks, spleens were isolated, enzymatically dissociated using Liberase Blendzyme III and dendritic cells isolated by autoMACS magnetic bead purification using pan-DC beads. Cell purity was measured by flow cytometry of CD11c expression and was >95% in each experiment. This purification scheme yielded a mixture of conventional CD11chi dendritic cells as well as PDCA1+ plasmacytoid cells, and the ratio of these two populations was consistent between experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction using Trizol. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile.
|
| Label |
Cy3
|
| Label protocol |
One µg total RNA from sample and reference were labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer
|
| |
|
| Channel 2 |
| Source name |
Common reference RNA, equal mixture of all 9 dendritic cell RNA study samples
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
sample type: reference
|
| Treatment protocol |
Cells were infected with H1N1 influenza A/Puerto Rico/8/34 at an MOI=10, stimulated with 30 ug/mL polyI:C, or cultured in media alone for 8 h (RPMI-1640, 10% heat-inactivated FBS, 2 mM L-glutamine, penicillin, streptomycin, 100 μM β-mercaptoethanol).
|
| Growth protocol |
C57Bl/6 mice were injected subcutaneously between the shoulder blades with 6x106 B6-melanoma cells expressing Flt3L to expand the dendritic cell compartment. After 2 weeks, spleens were isolated, enzymatically dissociated using Liberase Blendzyme III and dendritic cells isolated by autoMACS magnetic bead purification using pan-DC beads. Cell purity was measured by flow cytometry of CD11c expression and was >95% in each experiment. This purification scheme yielded a mixture of conventional CD11chi dendritic cells as well as PDCA1+ plasmacytoid cells, and the ratio of these two populations was consistent between experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction using Trizol. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile.
|
| Label |
Cy5
|
| Label protocol |
One µg total RNA from sample and reference were labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer
|
| |
|
| |
| Hybridization protocol |
The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY™ LNA array version 10.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for all species registered in the miRBASE version 11.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes.
|
| Scan protocol |
The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA).
|
| Description |
Biological replicate 1 of 3, PolyI:C 30 ug/mL, 8 hours.
|
| Data processing |
The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. The genes were selected based on two-tailed T-test calculated between the two sample groups with p-values lower than 0.001, and some of the genes were selected with further Bonferroni correction.
|
| |
|
| Submission date |
Mar 06, 2012 |
| Last update date |
Dec 05, 2012 |
| Contact name |
Aderem Lab |
| E-mail(s) |
[email protected]
|
| Organization name |
Seattle Children's Research Institute
|
| Lab |
Aderem Lab
|
| Street address |
307 Westlake Avenue North, Suite 500
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL7722 |
| Series (1) |
| GSE36316 |
Murine primary dendritic cells: Control vs. Influenza-infected vs. polyI:C stimulated. |
|
