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Sample GSM529517 Query DataSets for GSM529517
Status Public on Jun 28, 2010
Title ExvivoLung_VI14d_1
Sample type RNA
 
Source name Lung
Organism Mus musculus
Characteristics strain: BALB/c
tissue: Lung
treatment group: Vaccinated with BCG after 6 weeks and Infected with M. bovis after 14 days
Treatment protocol Five mice per group were sacrificed to evaluate the immune response of splenic lymphocytes. Spleen cells were prepared by passage through a 40μm cell strainer into DMEM supplemented with 10% (v/v) foetal calf serum (FCS) and antibiotics (100 U/ml penicillin and 100μg/ml streptomycin) (Gibco, UK) Following washing at 300 x g for 10 minutes cells were suspended at 5 x 10 to 6 /ml for assay. Lung cells were isolated by follow. Briefly, thoracic cavities were opened and gently inject sterile HBSS into right ventricle to perfuse lungs. Mince lungs were resuspended into digestion media DMEM supplemented plus 10 U/ml DNAse II (Sigma) and 150 U/ml collagenase type I (Gibco) and incubated for 1 hour at 37 ˚C and 200 rpm. After digestion lungs were prepared by passage through 100 μm cell strainer into DMEM. Following washing at 1600 rpm for 5 minutes, cells were poured into another tube through a 40 μm cell strainer, washed and used at 5 x 10 to 5.
Growth protocol Two groups of 20 mice each were immunised by a single intradermal injection of 2 x 105 CFU of M. bovis BCG (Vaccinated), or Hanks buffered salt solution (HBSS) (Unvaccinated). Six weeks later 5 mice from each group were euthanized for immunological analyses and the remaining mice from each group were challenged with approx 600 CFU M. bovis via the intranasal route. At days 3 and 14 post challenge five mice per group were euthanized and spleens and lungs harvested.
Extracted molecule total RNA
Extraction protocol RNA was isolated using standard RNA extraction protocols (Trizol). RNA samples were quality-checked via the Agilent 2100 Bioanalyzer platform
Label Cy3
Label protocol For the linear T7-based amplification step, 0.5 μg of each total RNA samples was used. To produce Cy3-labeled cDNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit following the manufacturer’s protocol. Yields of cDNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer.
 
Hybridization protocol The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit. Briefly, 1.65 μg Cy3-labeled fragmented cDNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to Agilent Whole Mouse Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6x SSPE buffer (3.6M NaCl, 0.2M NaH2PO4, 0.02M EDTA pH 7.4) contaning 0.005% N-lauroylsarcosine for 1 min at room termperature followed by a second wash with preheated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 seconds.
Scan protocol Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System. The Agilent Feature Software (FES) was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers and calculates statistical confidences.
Description Vaccinated with BCG after 6 weeks and Infected with M. bovis after 14 days
Data processing For determination of differential gene expression FES derived output data files were further analyzed using GeneSpring GX 11 (Agilent). The default normalization for Agilent one-color data in the GeneSpring GX program is quantile normalization following Agilent’s guidelines. After normalization we decided to focus on those genes that reliably change their expression, then we filtered the microarrays following three conditions: 1) Filter by value. Genes that do not have normalized signal intensity values of more than -0.5 and 0.5 were disregarded. 2) Filter by flags. All the genes with flags values present in at least 100% of the values in any 1 out of the 6 conditions were considered. 3) Filter by percentile. All the genes with normalized signal intensity values between 25 and 100 in any 1 out of the 6 conditions were also considered. Finally, all the genes in coincidence between filtering by flags group and filtering by percentile group were kept for statistical analysis. After filtering, parametric test (analysis of variance) was applied to compare mean expression levels in each analysis. Data were considered significant when the Benjamini Hochberg false discovery rate (FDR) for the comparison under analysis was <0.05, and the significance level was <0.05. In order to focus on highly regulated genes, we also restricted the majority of the analysis to genes with changes in expression levels of at least 1.7-fold change in all the conditions.
 
Submission date Mar 31, 2010
Last update date Jun 28, 2010
Contact name ELIHU Aranday-Cortes
Organization name Central Veterinary Laboratories Agency-Weybridge
Department SEB 4
Lab TB Research Group
Street address Woodham Lane, New Haw
City ADDLESTON
State/province SURREY
ZIP/Postal code KT153NB
Country United Kingdom
 
Platform ID GPL7202
Series (1)
GSE21149 Mycobacterium bovis-BCG vaccination induces a specific pulmonary transcriptome biosignature in mice

Data table header descriptions
ID_REF
VALUE GeneSpring normalized signal intensity

Data table
ID_REF VALUE
A_52_P616356 3.580515
A_52_P403405 4.9179974
A_52_P819156 2.8961067
A_51_P331831 0.19777012
A_51_P430630 3.9148335
A_52_P502357 6.883888
A_52_P299964 4.140954
A_51_P356389 4.67243
A_52_P684402 1.8858423
A_51_P414208 5.7316484
A_51_P280918 1.3302455
A_52_P613688 0.13595581
A_52_P258194 1.9705119
A_52_P229271 -0.47838402
A_52_P579519 -1.1019411
A_52_P979997 -0.005602002
A_52_P453864 -1.2343879
A_52_P655842 -0.395689
A_51_P282374 2.98E-04
A_52_P176013 1.2347245

Total number of rows: 41196

Table truncated, full table size 968 Kbytes.




Supplementary file Size Download File type/resource
GSM529517.txt.gz 8.0 Mb (ftp)(http) TXT
Processed data included within Sample table

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