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Sample GSM202029 Query DataSets for GSM202029
Status Public on Jan 01, 2010
Title RAW 264.7 – infected with Brucella abortus for 4hrs – replicate date 04192001b
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.
 
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) splitter@svm.vetmed.wisc.edu, angelajmathison@uwalumni.com
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

Data table header descriptions
ID_REF
VALUE Average Difference, the relative indicator of transcript level, calculated using MAS5.0 (see Affymetrix Literature)
ABS_CALL Absolute call of presence (P), absence (A) or marginal (M) generated by an Affymetrix decision matrix for each gene

Data table
ID_REF VALUE ABS_CALL
AFFX-MurIL2_at -618.3 A
AFFX-MurIL10_at -38.1 A
AFFX-MurIL4_at -6.9 A
AFFX-MurFAS_at 1175.9 A
AFFX-BioB-5_at 21507 P
AFFX-BioB-M_at 30571.4 P
AFFX-BioB-3_at 16816.1 P
AFFX-BioC-5_at 47845.5 P
AFFX-BioC-3_at 47436.9 P
AFFX-BioDn-5_at 57535.9 P
AFFX-BioDn-3_at 209605.5 P
AFFX-CreX-5_at 284197.8 P
AFFX-CreX-3_at 270186.8 P
AFFX-BioB-5_st 1858.8 A
AFFX-BioB-M_st 857.3 A
AFFX-BioB-3_st -668.5 A
AFFX-BioC-5_st -1158.2 A
AFFX-BioC-3_st -355.2 A
AFFX-BioDn-5_st 7491.4 P
AFFX-BioDn-3_st 1846.1 A

Total number of rows: 12654

Table truncated, full table size 215 Kbytes.




Supplementary file Size Download File type/resource
GSM202029.CEL.gz 3.1 Mb (ftp)(http) CEL
Processed data included within Sample table

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