|
| Status |
Public on Aug 01, 2010 |
| Title |
blood A/J 0hours rep4 |
| Sample type |
RNA |
| |
|
| Source name |
blood A/J mouse strain NOT infected with S. aureus
|
| Organism |
Mus musculus |
| Characteristics |
strain: A/J
infection: none
time point: 0
|
| 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. For experiments involving RNA analysis, blood was collected by intracardiac puncture and stored in RNAlater at -20oC.
|
| Growth protocol |
S. aureus strain Sanger 476 was used in this study. For preparation of S. aureus for injection, an overnight bacterial culture of S. aureus was diluted with fresh tryptic soy broth (TSB) and incubated (37°C) with aeration to log-phase growth (optical density at wavelength 600 nm (O.D.600) of ~1.0) (Rice 2003). S. aureus was harvested by centrifugation, rinsed, and resuspended in saline.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using the Mouse RiboPure Blood RNA isolation kit (Ambion) following the manufacturer’s instruction. Globin mRNA was removed from whole blood RNA samples using the Globinclear kit (Ambion).
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 0.5 ug total RNA (Affymetrix).
|
| |
|
| Hybridization protocol |
Labeled cDNA was hybridized to the arrays for 16 hours at 45°C following the manufacturer’s instruction. The 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.
|
| Scan protocol |
Labeled gene chips were scanned using an Affymetrix Genechip Scanner 7G (Affymetrix).
|
| Description |
none
|
| Data processing |
Preprocessing was conducted using the Robust Multichip Analysis (RMA) (PMID: 12925520) implementation in the Bioconductor “affy” package (http://www.bioconductor.org/), with an additional step to account for differences in probe hybridization resulting from single nucleotide polymorphisms (SNP) between A/J and C57BL/6J mice. The additional step is referred to as SNP masking (PMID:17762873) and is applied after background correction and quantile-quantile normalization but prior to the determination of probeset expression values. Genomic locations hybridized by each probe were obtained from the Ensembl database (http://www.ensembl.org/index.html), and these genomic locations were compared to the locations of SNPs for which A/J and C57BL/6J mice have different alleles. Probes that hybridize to such SNPs within the target transcripts were excluded from the determination of probeset expression values. Statistically significant differences in expression between A/J and C57BL/6J mice were determined using a standard ANOVA with model terms for batch, strain, and time post-infection, with uninfected mice coded as 0 hours post-infection.
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| |
|
| Submission date |
Dec 28, 2009 |
| Last update date |
Jul 27, 2010 |
| Contact name |
Lindsay Cowell |
| E-mail(s) |
lgcowell@duke.edu
|
| Organization name |
Duke University
|
| Street address |
2424 Erwin Road
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27705 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE19668 |
Genetic Determinants for Susceptibility to Staphylococcus aureus Infection in A/J and C57BL/6J |
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