|
| Status |
Public on Jan 01, 2010 |
| Title |
RAW 264.7 – infected with Brucella abortus for 4hrs – replicate date 07252001 |
| Sample type |
RNA |
| |
|
| Source name |
RAW 264.7, infected with Brucella abortus, 4 hours
|
| Organism |
Mus musculus |
| Characteristics |
designation: RAW 264.7 macrophages
atcc: TIB-71
growth properties: adherent
tissue: Abelson murine leukemia virus-induced tumor
antigen expression: H-2d
strain: BALB/c mice
bacteria: Brucella abortus, Gram negative, facultative intracellular pathogen
strain: S2308
|
| Biomaterial provider |
American Type Culture Collection (ATCC), G. Splitter Stock Cultures
|
| Treatment protocol |
RAW 264.7 cells for microarray were plated in 75cm2 flasks 12-24hr prior to infection, in supplemented RPMI 1640 without antibiotics at an approximate concentration of 6x 10^6 cells per flask. Macrophage cells were washed once with PBS prior to infection and 5 mL fresh media added. One mL of actively growing Brucella culture was added to the macrophages (MOI ~100:1). Infections were allowed to incubate for 4 hours at 37°C with 5% CO2. Cells were washed three times with PBS to remove extracellular bacteria and then cells were lysed for RNA collection (RNeasy Mini Kit (Qiagen, Valencia, CA)).
|
| Growth protocol |
RAW 264.7 (TIB-71, ATCC) mouse macrophage (Mφ) cell line was maintained at 37°C with 5% CO2 in RPMI 1640 (Invitrogen) supplemented with 10% Fetal Bovine Serum, 0.2mM L-glutamine, 100U penicillin/mL, 100ug streptomycin/mL (Sigma). Macrophages were subcultured as necessary by removing old media, washing with PBS, adding fresh media, scraping cells and diluting cells 1:3 to 1:20.
B. abortus was grown in 12- by 75-mm tubes on a shaker platform in BBL Brucella broth (BD Bioscience). Cultures for infection were grown at 37°C for 3 days prior to infection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from macrophage cultures utilizing RNeasy Mini Kit (Qiagen) according to manufacturer’s protocol with the following modifications. Cells were lysed in RLT and then centrifuged to remove intact bacterial cells. DNase treated RNA was converted to double stranded cDNA using a synthesis kit (Gibco) except that T-7-(dT)24 oligomer (Genset Corp.) was used. The double stranded cDNA was isolated with the GeneChip® Sample cleanup module.
|
| Label |
biotin
|
| Label protocol |
Target RNA was prepared according to protocols in the Affymetrix GeneChip® Expression Analysis technical manual (Affymetrix, Santa Clara, CA). In vitro synthesis of biotin-labeled cRNA was completed with Enzo BioArray HighYield RNA Transcript Labeling Kit (Affymetrix). In vitro transcription clean up was completed over an RNeasy spin column (Qiagen). Labeled cRNA mixed with fragmentation buffer was incubated at 94°C for 35 minutes. Final RNA concentration in fragmentation buffer ranged from 0.5-1.1 μg/μL. Visual confirmation of isolation and fragmentation was completed with agarose gel electrophoresis.
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| |
|
| Hybridization protocol |
Labeled cRNA was hybridized to GeneChip® Murine Genome U74A microarrays (Affymetrix). GeneChip® hybridization, washing and scanning was performed by the University of WI –Biotechnology Center, Gene Expression Center (University of WI, Madison) utilizing all Affymetrix protocols and procedures. Washing and streptavidin-phycoerythrin staining was completed on a GeneChip Fluidics station 400 (Affymetrix).
|
| Scan protocol |
GeneChips were scanned with HP 2500 GeneArray Scanner and signal output analysis completed with MAS5.0 (Affymetrix).
|
| Description |
See other fields.
|
| Data processing |
Signal output analysis completed with U74A Mask Files and MAS5.0 (Affymetrix). For original analysis Eskra, et al. Infection and Immunity 71(3), 2003 the difference calls were assigned values and summed across the six comparisons.
|
| |
|
| Submission date |
Jun 18, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Gary Splitter |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
University of Wisconsin - Madison
|
| Department |
Pathobiological Sciences
|
| Lab |
Dr. Gary Splitter
|
| Street address |
1656 Linden Dr
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL32 |
| Series (2) |
| GSE8384 |
Microarray Analysis of mRNA Levels from RAW264.7 Macrophages Infected with Brucella abortus |
| GSE8403 |
RAW264.7 macrophages infected with Brucella abortus, B. melitensis, B. neotomae, and B. ovis |
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