|
| Status |
Public on Jan 01, 2009 |
| Title |
Sample_I2_BMDMs_infected_24h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
BALB/c mouse bone marrow-derived macrophages
|
| Organism |
Mus musculus |
| Characteristics |
Female Swiss nu/nu mice , between 8 and 12-week old were used for L. amazonensis strain LV79 (MPRO/BR/1972/M1841) propagation.
|
| Treatment protocol |
At t=24h, amastigotes were added at a multiplicity of 4 amastigotes per bone marrow-derived macrophage. Parasite-housing macrophages (>98%) were cultured at 34°C for 24h.
|
| Growth protocol |
Bone marrow cell suspensions recovered from tibias and femurs of BALB/c mice were suspended in RPMI 1640 medium (Seromed) supplemented with 10% FCS, antibiotics and 20% L-929 fibroblast-conditioned medium. Cells were distributed in bacteriologic Petri dishes (Greiner, Germany) and incubated at 37 °C in a 5% CO2 air atmosphere. Six days later, bone marrow-derived smoothly adherent macrophages (BMDMs) were washed with Dulbecco’s phosphate buffered solution (PBS) and resuspended in prewarmed 1% EDTA in Dulbecco’s PBS without Ca2+ and Mg2+ (Biochrom AG, Berlin, Germany). Recovered BMDMs were deposited in flat-bottom 24-well plates (Tanner, Switzerland) at a density of 5 * 10e-05 cells per well (reference time: t=0h).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction directly from living Macrophages(RNeasy+ Mini-Kit, Qiagen), RNA quality control and concentration were determined using the NanoDrop ND-1000 microspectrophotometer. RNA integrity was assessed using the Agilent-2100 Bioanalyzer (RNA Integrity Numbers ≥ 9/10).
|
| Label |
biotin
|
| Label protocol |
Affymetrix standard protocol:
www.affymetrix.com/support/technical/other/oncecyle_presentation.pdf
|
| |
|
| Hybridization protocol |
Affymetrix EukGE-WS2v5 Hybridization
|
| Scan protocol |
Standard Affymetrix protocol
|
| Description |
Leishmania RNA did not interfere with BALB/c mouse RNA hybridization onto GeneChips by increasing the fluorescence signal (data not shown). Indeed, FC values for a technical replicate of mouse RNA were comparable to those for mouse RNA “contaminated” with up to 10% of L. amazonensis RNA taking the “non-contaminated” mouse RNA as a reference (P-value ~ 0, one-sided paired Student’s t-test for FC increases).
|
| Data processing |
Data processing, background correction, normalization and signal quantification were carried out with GC-Robust Multiarray Analysis (GC-RMA) algorithm using all samples (I1, I2, UI1 and UI2).
Unreliable probe-sets called “Absent” by Affymetrix GeneChip Operating Software (GCOS, www.affymetrix.com/support/technical/manual/expression_manual.affx) for at least 3 out of 4 GeneChips were discarded from subsequent analyses, as well as probe-sets called “Absent” once within samples plus once within controls (total number of probe-sets filtered out: 25,994 out of 45,101, i.e. 57.64%; see online supplementary material).
Local pooled error (LPE) tests were performed to identify significant differences in gene expression between parasite-free and parasite-harboring MΦs.
Benjamini-Hochberg (BH) multiple-test correction was applied to control for the number of false positives with an adjusted 5% statistical significance threshold.
We used GC-RMA algorithm, LPE tests and BH multiple-test correction as implemented in Splus ArrayAnalyzer® module (www.insightful.com/products/s-plus_arrayanalyzer/).
A total of 1,248 probe-sets showed significant differential expression, and were input into Ingenuity Pathway Analysis software 5.5.1 (www.ingenuity.com) to perform a biological interaction network analysis.
|
| |
|
| Submission date |
May 19, 2008 |
| Last update date |
Aug 02, 2019 |
| Contact name |
Eric Prina |
| E-mail(s) |
eric.prina@pasteur.fr
|
| Phone |
+33 (0)140613514
|
| Organization name |
Institut Pasteur
|
| Department |
Parasitology and Mycology
|
| Lab |
IPPIC
|
| Street address |
25 rue du Dr Roux
|
| City |
Paris |
| ZIP/Postal code |
75724 |
| Country |
France |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE11497 |
Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes |
|
| Relations |
| Reanalyzed by |
GSE45704 |
| Reanalyzed by |
GSE119085 |
| Reanalyzed by |
GSE135324 |
