|Application:||Gene expression microarray analysis|
|Number of samples:||36|
|Release date:||Sep 20 2008|
|Last update date:||Mar 26 2012|
|Diseases:||Mycobacterium Infections, Nontuberculous, Tuberculosis, Bovine|
|Dataset link||Comparison of antigen stimulation response in Mycobacterium bovis-infected vs. control cattle using BOTL-5 microarray|
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.
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