 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 16, 2007 |
| Title |
Comparison of Mycobacterium bovis-infected cattle versus control non-infected cattle using the BOTL-5 microarray |
| Organism |
Bos taurus |
| Experiment type |
Expression profiling by array
|
| Summary |
Background: Bovine tuberculosis is an enduring disease of cattle that has significant repercussions for human health. The advent of high-throughput functional genomics technologies has facilitated large-scale analyses of the immune response to this disease that will ultimately lead to novel diagnostics and therapeutic targets. Analysis of mRNA abundance in peripheral blood mononuclear cells (PBMC) from six Mycobacterium bovis infected cattle and six non-infected controls was performed. A targeted immunospecific bovine cDNA microarray with duplicated spot features representing 1,391 genes was used to test the hypothesis that a novel gene expression profile may exist in M. bovis infected animals in vivo.
Results: In total, 378 gene features were differentially expressed at the P ≤ 0.05 level in bovine tuberculosis (BTB)-infected and control animals, of which 244 were expressed at lower levels (65%) in the infected group. Lower relative expression of key innate immune genes such as the Toll-like receptor 2 (TLR2) and TLR4 genes, lack of differential expression of indicator adaptive immune gene transcripts (IFNG, IL2, IL4), and lower BOLA major histocompatability complex – class I (BOLA) and class II (BOLA-DRA) gene expression was consistent with innate immune gene repression in the BTB-infected animals. Supervised hierarchical cluster analysis and class prediction validation identified a panel of 15 genes predictive of disease status and selected gene transcripts were validated (n = 8 per group) by real time quantitative reverse transcription PCR.
Conclusion: These results suggest that large-scale expression profiling can identify gene signatures of disease in peripheral blood that can be used to classify animals on the basis of in vivo infection, in the absence of exogenous antigenic stimulation.
Keywords: Disease state analysis
|
| |
|
| Overall design |
Experimental animals
Sixteen cattle were used for this study. The eight infected animals were chosen from herds with a recent history of chronic infection with M. bovis. The animals were selected on the basis of the skin-fold thickness response to bovine and avian tuberculin in the single intradermal comparative tuberculin test (SICTT). The SICTT reactor animals were selected where the skin-fold thickness response to PPD-bovine exceeded that of PPD-avian by at least 12 mm. All of these animals were also measured positive in a whole blood IFN-g assay. The cattle were confirmed positive for tuberculosis following detailed post-mortem pathological examination and/or culture. Bronchial, mediastinal, submandibular, retropharyngeal, mesenteric and hepatic lymph nodes and lungs were examined macroscopically for tuberculosis lesions. Suspected lesions were cultured on Stonebrinks and Lowenstein-Jensen media at 37°C for eight weeks to detect M. bovis. The eight non-infected control animals were selected from a herd without a recent history of tuberculosis and were SICTT and IFN-g test negative.
Blood sampling and analysis
400 ml of blood was collected from each animal in sterile heparinised bottles. Five ml of blood was used for haematological analysis using an Abbott CELL-DYN 3500R automated haematology analyzer (Abbott Laboratories). Leukocyte cell population subsets were compared between infected and control groups (n = 8) using Student’s t-test.
PBMC separation, RNA extraction and quality control
PBMC were isolated using the Percoll™ gradient method with a standard protocol [48]. PBMC were seeded at 10^7 per culture plate and cultured in RPMI 1640 medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamicin. All PBMC samples were cultured overnight at 37°C in 5% CO2. Overnight culture was carried out to minimise noise in gene expression measurements potentially introduced by the mechanical disruption of cells associated with PBMC isolation. Residual cells not seeded for culture were immediately suspended in 3 ml TriReagent® (Molecular Research Centre Inc.) and frozen in 1.5 ml cryotubes at -80°C for use later as a common reference RNA (CRR) pool. Total RNA was extracted using a combined TriReagent®, DNase treatment and Qiagen RNeasy® method (Qiagen Ltd.) according to the manufacturers’ instructions. The integrity and stability of RNA samples is crucial for gene expression analyses using microarray technology; therefore, RNA yield and quality were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). The two-step method for RNA extraction described above was found to produce RNA of high yield and quality (ratios of 18S to 28S ribosomal RNA averaged > 1.6).
Microarray experimental design
The 3,888 feature BOTL-5 immunogenetic cDNA microarray system used has been described previously [49]. Technical information with gene content and sequence information for the BOTL-5 array can be downloaded from the ‘Links’ section on the MSU Center for Animal Functional Genomics website (http://www.nbfgc.msu.edu). The immunobiology-targeted BOTL-5 array contains 1,391 genes or ESTs spotted in duplicate with multiple additional control features (blank spots, negative spots, housekeeping genes) and is an expanded version of the BOTL-4 array described previously. A reference design was used for microarray hybridizations, such that all RNA samples were labelled using Cy3 and co-hybridized with Cy5 labelled CRR pool. It was hypothesized that the CRR pool would display similar mRNA expression levels and gene coverage as the target samples, therefore allowing accurate and consistent comparison of gene expression data without arbitrarily pairing animals from the two groups. The CRR pool contained equal amounts of total RNA from the treated and control animal groups. Twelve arrays were hybridized in total, representing six individual animals from each treatment group.
|
| |
|
| Contributor(s) |
Meade KG, Gormley E, Doyle MB, Fitzsimons T, O’Farrelly C, Costello E, Keane J, Zhao Y, MacHugh DE |
| Citation(s) |
17974019 |
| |
| Submission date |
Aug 23, 2007 |
| Last update date |
Mar 17, 2012 |
| Contact name |
David E MacHugh |
| E-mail(s) |
david.machugh@ucd.ie
|
| Phone |
+353-1-7166256
|
| Organization name |
University College Dublin
|
| Department |
College of Agriculture, Food Science and Veterinary Medicine
|
| Lab |
Animal Genomics Laboratory
|
| Street address |
University College Dublin, Belfield
|
| City |
Dublin |
| ZIP/Postal code |
D4 |
| Country |
Ireland |
| |
|
| Platforms (1) |
| GPL5751 |
Bovine Total Leukocyte cDNA microarray: Fifth generation microarray |
|
| Samples (12)
|
|
| Relations |
| BioProject |
PRJNA102213 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE8857_RAW.tar |
4.4 Mb |
(http)(custom) |
TAR (of GPR) |
| Processed data included within Sample table |
|
|
|
|
 |
Less...
More...