|
| Status |
Public on Jun 28, 2010 |
| Title |
ExvivoLung_VI14d_5 |
| 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.
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| |
|
| 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 |
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