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| Status |
Public on Jan 11, 2013 |
| Title |
blood CD1 24hrs Ecoli rep10 batch 9 batch 9 1545_4571_25352_Ecoli_10 |
| Sample type |
RNA |
| |
|
| Source name |
blood CD1 mouse strain blood taken 24 hours after infection with E. coli
|
| Organism |
Mus musculus |
| Characteristics |
host strain: CD1
host gender: male
host age (yr): 0.50-0.66
pathogen: Escherichia coli
bacterial strain: O18:K1:H7
pathogen dosage cfu/g: 6.00E+04
inoculum route: peritoneum
anatomic site of infection: -
infection status: infected
time after infection (h): 24
replicate number: 10
experimental batch: 9
|
| 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.
|
| Growth protocol |
One methicillin-susceptible S. aureus strain (Sanger 476) and three methicillin-resistant S. aureus strains (USA100, USA300, and MW2) were 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). E. coli O18:K1:H7 was grown at 30°C overnight in Luria Bertani broth (Miller et al., 1972). Cultures were then diluted with fresh medium and grown for an additional 1 to 2 hours. Upon reaching log phase, cells were harvested by centrifugation, washed, and resuspended in 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. Microarray data was analyzed in two steps following the analysis strategy previously outlined and utilized (Zaas et al., 2010). First, a Bayesian sparse factor model was fit to the expression data without regard to phenotype (Carvalho et al., 2008; Wang et al., 2007). Second, factors were then used to build a probit regression with variable selection model (Hans et al., 2007) trained to identify S. aureus infection.
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| |
|
| Submission date |
Oct 31, 2011 |
| Last update date |
Jan 11, 2013 |
| 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) |
| GSE33341 |
Gene Expression-Based Classifiers Identify Staphylococcus aureus Infection in Mice and Humans |
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