|
| Status |
Public on Jan 08, 2009 |
| Title |
macIL-10tg Mtb d42 2 |
| Sample type |
RNA |
| |
|
| Source name |
lung RNA; day 42 after Mtb infection
|
| Organism |
Mus musculus |
| Characteristics |
Strain: macIL-10tg mice; tissue: lung; infected with M. tuberculosis for 42 days
|
| Treatment protocol |
Mtb (H37Rv) were grown in Middlebrook 7H9 broth supplemented with Middlebrook OADC enrichment medium 0.002 % glycerol, and 0.05 % Tween 80. Midlog phase cultures were harvested, aliquoted, and frozen at –80°C. After thawing, viable cell counts were determined by plating serial dilutions of the cultures on Middlebrook 7H10 agar plates followed by incubation at 37°C. Before infection of experimental animals, stock solutions of Mtb were diluted in sterile distilled water and pulmonary infection was performed using an inhalation exposure system. To infect mice with a low dose of 100 CFU/lung, animals were exposed for 40 min to an aerosol generated by nebulizing approximately 5.5 ml of a suspension containing 10exp7 live bacteria. Inoculum size was checked 24 h after infection by determining the bacterial load in the lung of infected mice.
|
| Growth protocol |
Transgenic mice (macIL-10tg mice) specifically expressing IL-10 in macrophages under control of the human CD68 promoter were bred under specific-pathogen-free conditions at the Technical University of Munich. macIL-10tg mice were on a FVB genetic background and transgene-negative littermates were used as controls (FVB). For experiments, mice were maintained under barrier conditions in the BSL 3 facility at the Research Center Borstel in individually ventilated cages.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Before and at different time points after aerosol infection with Mtb, weighed lung samples were homogenized in 5 ml of 4 M guanidinium-isothiocyanate buffer and total RNA was extracted by acid phenol extraction.
|
| Label |
biotin
|
| Label protocol |
Affymetrix GeneChips were used for genome-wide expression analysis of the impact of IL-10 overexpression during pulmonary Mtb infection. Tissue from individual mice was processed as biological duplicates (untreated controls and day 25 after infection) and triplicates (day 42 after infection). Per sample, 3 µg total lung RNA was processed using the GeneChip Expression 3’ Amplification One-Cycle Target Labeling Kit according to the manufacterer’s instruction.
|
| |
|
| Hybridization protocol |
Biotinylated cRNA was hybridized on MOE430A 2.0 GeneChips, that were stained, washed and scanned following standard procedures.
|
| Scan protocol |
Standard Affymetrix protocol
|
| Description |
Gene expression data from lung of mice 42 days after M. tuberculosis infection
|
| Data processing |
CEL files were processed for global normalization and generation of expression values using the robust multi-array average algorithm (RMA) (ref.: Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185-93 (2003)).
|
| |
|
| Submission date |
Jan 07, 2009 |
| Last update date |
Jun 03, 2013 |
| Contact name |
Roland Lang |
| E-mail(s) |
roland.lang@uk-erlangen.de
|
| Organization name |
University Hospital Erlangen
|
| Street address |
Wasserturmstr. 3-5
|
| City |
Erlangen |
| ZIP/Postal code |
91054 |
| Country |
Germany |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE14316 |
Impact of IL-10 pulmonary gene expression in mouse M. tuberculosis infection |
|
