|
| Status |
Public on Oct 24, 2011 |
| Title |
kidney-infected with S aureus HG001 ΔsigB-BR1-1 |
| Sample type |
RNA |
| |
|
| Source name |
murine kidney d4 post infection with HG001 ΔsigB
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
gender: female
tissue: kidney
|
| Treatment protocol |
Female BALB/c mice (Charles River) were i.v. infected in two biological replicates with 5E+07 to 7E+07 colony-forming units (CFUs) of S. aureus HG001 or 5E+07 to 8E+07 CFUs of S. aureus HG001 ΔsigB. As a control, another group of mice received an injection of 100 µl physiological saline solution. Mice were sacrificed after 4 (1. biological replicate) or 5 days (2. biological replicate).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Kidneys were explanted and immediately frozen in liquid nitrogen. Afterwards, both kidneys of the mice were homogenized using a mortar filled with liquid nitrogen. Kidney tissue was mixed with 0.5 ml TRIzol (Invitrogen) under liquid nitrogen and then mechanically disintegrated at 2600 rpm for 2 min in a bead mill (Mikrodismembrator S, B. Braun Biotech International GmbH). After addition of further 0.5 ml TRIzol and incubation for 10 min at room temperature, RNA isolation was performed according to the manufacturer’s protocol with the following modifications: i) overnight RNA precipitation at -20°C; ii) pelleting the RNA precipitate for 30 min at 20000 x g. The resulting RNA was DNase treated (6.8 Kunitz units) for 10 min at room temperature (QIAGEN GmbH) and afterwards purified using a RNA Clean-Up and Concentration Kit (Norgen Biotek Corp.). RNA concentrations were determined photometrically (NanoDrop Technologies), and the quality was controlled with an Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
The host RNA expression profile was analyzed on GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) using the Whole Transcript (WT) Sense Target Labeling and Control reagents according to the manufacturer’s instructions.
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| |
|
| Hybridization protocol |
The host RNA expression profile was analyzed on GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) using the Whole Transcript (WT) Sense Target Labeling and Control reagents according to the manufacturer’s instructions. Arrays were washed and stained in a GeneChip FluidicsStation 450 and scanned with a GeneChip Scanner 3000 (Affymetrix).
|
| Scan protocol |
Arrays were washed and stained in a GeneChip FluidicsStation 450 and scanned with a GeneChip Scanner 3000 (Affymetrix).
|
| Description |
BR1_inf sigB
|
| Data processing |
The array image files (CEL) were first quality controlled in the Expression Console software (Affymetrix) and then imported into the Rosetta Resolver software (Rosetta Biosoftware) for data analysis. Signals were generated and normalized using the RMA algorithm. Groups were compared in log-transformed space using error-weighted one-way ANOVA with Benjamini-Hochberg False Discovery Rate multiple testing correction and p* < 0.01 was regarded as significant. The control sequences and sequences that were not considered expressed on all arrays belonging to the selected group comparison (with p > 0.01 on intensity profile level in Rosetta Resolver software) were not included in statistical testing. Afterwards, sequence sets were translated into EntrezGene records using the Rosetta Resolver annotation of December 2009. Two criteria were used for definition of differential expression: Significance in ANOVA statistical testing and a minimal absolute fold change of 2. Further criteria only applied to the comparison of infection with non-infected controls: Significance in ANOVA statistical testing and a minimal absolute fold change of 2 in at least one of two comparisons (“infection with HG001” vs. “control”; “infection with HG001 ΔsigB” vs. “control”). Genes significant in ANOVA but with a minimal absolute fold change of only 1.5 in at least one of two comparisons were considered to be regulated by trend.
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| |
|
| Submission date |
Apr 12, 2011 |
| Last update date |
Oct 25, 2011 |
| Contact name |
Maren Depke |
| Organization name |
Ernst-Moritz-Arndt-University Greifswald
|
| Department |
Functional Genomics
|
| Street address |
Friedrich-Ludwig-Jahn-Straße 15a
|
| City |
Greifswald |
| ZIP/Postal code |
17489 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE28540 |
Activity of SigB Modulates Virulence Gene Expression in a Murine Staphylococcus aureus Infection Model but Does Not Influence the Host Kidney Gene Expression |
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