|
| Status |
Public on Jun 05, 2014 |
| Title |
blood at T0, vancomycin treated, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
mouse blood after vancomycin treatment (i.v.)
|
| Organism |
Mus musculus |
| Characteristics |
inbred strain: A/J
drug treatment (linezolid/vancomycin/no): vancomycin
time (0h/2h/24h post infection): 0
replicate: 2
host gender: male
age: 8-10 weeks
|
| Treatment protocol |
To mimic the natural course of S. aureus infection in humans, which typically arises from a primary focus of infection and disseminates to other sites, we employed an intraperitoneal (i.p.) route of infection in our animal model. Briefly, blood from 5 individual mice from A/J will be collected by intracardiac puncture after infection with USA300, and 24h post-treatment of linezolid vs vancomycin (i.v.) and stored in RNAlater at -20oC
|
| Growth protocol |
One methicillin-resistant S. aureus strain (USA300) was used. Overnight S. aureus cultures were inoculated into fresh tryptic soy broth and incubated aerobically at 30°C to log-phase growth (optical density 600nm of ~1.0) (Rice et al., 2003). Cells were harvested by centrifugation, rinsed, and resuspended in phosphate-buffered saline (PBS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from mouse blood using the Mouse RiboPure Blood RNA kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Globin mRNA was removed from whole blood RNA using the Globinclear kit (Ambion, Austin, TX). All samples passed the quality criteria of the Agilent Bioanalyzer and were used for microarray analysis. Since the total RNA yield of many samples was low, one round of linear amplification was performed for all samples using the MessageAmp Premier kit (Ambion, Austin, TX).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 0.5 ug total RNA (Affymetrix).
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| |
|
| Hybridization protocol |
Biotin-labeled cDNA was hybridized to the arrays for 16 hours at 45°C according to the manufacturer’s instruction. Arrays were then washed and labeled with streptavidinphycoerythrin (strep-PE), and the signal was amplified using biotinylated antistreptavidin followed by another round of staining with strep-PE. These steps were performed on the Affymetrix fluidics station according to the recommended protocol.
|
| Scan protocol |
Amplification and microarray hybridization were performed at the Duke University Microarray Core. Labeled gene chips were scanned using an Affymetrix Genechip Scanner 7G (Santa Clara, CA).
|
| Data processing |
Data processing was conducted using the Robust Multichip Average (RMA) generated by Affymetrix Expression Console software. Gene expression data were imported into Partek Genomics Suite 6.5 (Partek, St Louis, Mo) as CEL files using default parameters. Raw data were preprocessed, including background correction, normalization, and summarization using robust multiarray average analysis, and expression data were log2 transformed. Differential expression analysis for the whole blood cells was performed using 1-way ANOVA (either infection status or drug treatment alone). Gene lists were created using a cutoff of P < .05, 2-fold change, although second-level analysis using a false-discovery rate of <0.05, 2-fold change was also performed.
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| |
|
| Submission date |
Jun 06, 2012 |
| Last update date |
Jun 05, 2014 |
| Contact name |
Sun Hee Ahn |
| Organization name |
Duke University
|
| Department |
Medicine
|
| Lab |
Infectious Diseases (Staphylococcal Disease)
|
| Street address |
Stead Bldg, RM 1546A
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE38531 |
Expression data in Staphylococcus aureus-infected mice with linezolid and vancomycin treatment |
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