|
| Status |
Public on Nov 08, 2012 |
| Title |
Human Dendritic Cell L. major 24hrs biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Human Dendritic Cell L. major 24hrs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: monocyte-derived dendritic cells
leishmania major infected: yes
time point: 24 hrs
|
| Treatment protocol |
L. major parasites were maintained in complete M199 media (supplemented with 20% FBS). Metacyclic promastigotes were isolated using a ficol density gradient protocol and opsonized by treatment with 5% human serum for 30 min at 37°C. Metacyclic promastigotes were utilized for infection of the monocyte-derived human dendritic cells
|
| Growth protocol |
Monocyte-derived human dendritic cells were maintained in RPMI-complete media (10% FBS, 2mM l-glutamine 100U/ml, 1% penicillin/streptomycin) at 37°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Uninfected and L. major infected samples were harvested at 2, 4, 8, or 24hrs post infection. Total RNA was extracted from each sample utilzing an RNEasy kit according to manufacturer's protocols (Qiagen, Valencia, CA)
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNAs were prepared according to standard Affymetrix protocol from 2μg of total RNA according to manufacturer's protocols (Affymetrix, Santa Clara, CA)
|
| |
|
| Hybridization protocol |
Purified cRNA was fragmented in 5X fragmentation buffer (200mM Tris-Acetate, pH8.1, 500mM potassium acetate, 150mM magnesium acetate) at 94ºC for 35 min prior to cRNA chip hybridization. Gene chips were washed and stained utilizing the Affymetrix Fluidics Station 400
|
| Scan protocol |
Gene chips were scanned with the Gene Chip Scanner 3000 (Affymetrix, Santa Clara, CA)
|
| Description |
Gene expression data from human monocyte derived dendritic cells, L. major infected, 24hrs
|
| Data processing |
The Affymetrix data files (.cel) were processed using Bioconductor software package (http://www.bioconductor.org/, Seattle, WA) [13]. Probe set signal intensities were subjected to background correction by GCRMA (Genespring GX 7.0) normalization across all 15 chips. The mean fluorescence intensity was derived from a log2 transformation of the raw data. Probe sets with “present” call in at least 7 of the 15 chips were retained, which filtered our list of 54,000 probe sets down to ~14,000. Statistical significance was determined using a one-way ANOVA and Benjamini-Yekutieli multiple correction test to control false discovery rates. A final set of 848 candidate probe set were identified as significantly expressed in at least one time point compared to non-infected samples (p<0.05).
|
| |
|
| Submission date |
Nov 06, 2012 |
| Last update date |
Nov 08, 2012 |
| Contact name |
Michelle A Favila |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Notre Dame
|
| Department |
Biology
|
| Lab |
Mary Ann McDowell
|
| Street address |
210 Galvin Life Science Center
|
| City |
Notre Dame |
| State/province |
IN |
| ZIP/Postal code |
46556 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE42088 |
Expression data from Leishmania major infected human dendritic cells |
|
