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Series GSE12835 Query DataSets for GSE12835
Status Public on Sep 20, 2008
Title Comparison of antigen stimulation response in Mycobacterium bovis-infected vs. control cattle using BOTL-5 microarray
Organism Bos taurus
Experiment type Expression profiling by array
Summary Bovine tuberculosis (BTB) caused by Mycobacterium bovis continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying chronic mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC) from BTB-infected animals are repressed in vivo in the absence of exogenous stimulation. In the present study, functional genomics methods were used to examine the immune response of PBMC from BTB-infected (n = 6) and healthy control (n = 6) cattle to stimulation with bovine purified protein derivative (PPDb) in vitro. PBMC were harvested before, and at 3h and 12h post in vitro stimulation with PPDb. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5) with 4,800 spot features representing 1,391 genes
 
Overall design Experimental animals and infection status
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 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-assay [ref.52]. 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 [ref.53]. The eight non-infected control animals were selected from a herd without a recent history of tuberculosis and were SICTT and IFN-gamma 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 and leukocyte cell population subsets were compared between infected and control groups as previously described [ref.24].
PBMC separation, culture, RNA extraction and quality control
PBMC were isolated using the Percoll gradient method as previously described [ref.54].PBMC were seeded at 10^7 per flask and cultured in RPMI 1640 culture medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamicin. A total of 48 tissue culture flasks represented 16 individual PBMC samples (BTB-infected [n = 8] and healthy control [n = 8]) per time point at 0h (pre-stimulation), 3h and 12h post-stimulation with PPDb. All PBMC samples were cultured overnight at 37°C in 5% CO2 to minimize noise in gene expression measurements potentially introduced by the mechanical disruption of cells associated with PBMC isolation. After cells were harvested for time point 0, remaining flasks were stimulated using 50 ug/ml PPD-bovine and incubated for 3h and 12h at 37°C in 5% CO2. Residual cells not seeded for culture in either treatment were immediately dissolved in 3 ml TriReagent (Molecular Research Centre Inc., Cincinnati, OH) and frozen in 1.5 ml cryotubes at -80°C for use as a common reference RNA (CRR) pool. Total RNA was extracted from PBMC harvested after 3h and 12h post-stimulation using a combined TriReagent, DNase treatment and Qiagen RNeasy method (Qiagen Ltd., Crawley, UK) according to the manufacturer 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 Ireland Ltd., Dublin, Ireland). 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 microarray system used has been described previously [ref.55]. The NCBI GEO platform accession for the BOTL-5 microarray is GPL5751. The immunobiology-targeted BOTL-5 array contains 1,391 genes or ESTs spotted in duplicate with multiple additional control features (blank spots, negative spots, housekeeper genes) and is an expanded version of the BOTL-4 array described previously by our group [refs.22, 56]. A reference design was used for microarray hybridizations, such that all RNA samples were labelled using Cy3 and co-hybridized with Cy5 labelled common reference RNA (CRR) pool as described previously [ref.24]. Thirty-six arrays were hybridized in total, representing six individual animal PBMC samples from each treatment group pre-stimulation, and at 3h and 12h post stimulation with PPDb, as shown in Fig. 1. It was hypothesized that the CRR pool would display similar mRNA expression levels and gene coverage as the target samples, therefore allowing flexible, accurate and consistent comparison of gene expression data across a time course without arbitrarily pairing animals from different groups [ref.57]. The CRR pool contained equal amounts of total RNA from the treated and control animal groups.
 
Contributor(s) Meade KG, Gormley E, O’Farrelly C, Park SD, Costello E, Keane J, Zhao Y, MacHugh DE
Citation(s) 18823559
Submission date Sep 18, 2008
Last update date Mar 26, 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 (36)
GSM220190 PBMC-CRR-vs-control-1
GSM220192 PBMC-CRR-vs-control-2
GSM220193 PBMC-CRR-vs-control-3
Relations
BioProject PRJNA156797

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE12835_RAW.tar 13.3 Mb (http)(custom) TAR (of GPR)
Processed data included within Sample table
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