Computational protocol: Global Gene Expression and Systems Biology Analysis of Bovine Monocyte-Derived Macrophages in Response to In Vitro Challenge with Mycobacterium bovis

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Protocol publication

[…] Affymetrix® GeneChip® Bovine Genome Array data were analysed using the BioConductor software project (http://www.bioconductor.org) contained within the R statistical environment (http://www.r-project.org). Quality control analysis was performed on all 49 microarrays using the Simpleaffy software package contained within BioConductor. Normalisation of raw data was performed using the Factor Analysis for Robust Microarray Summarization (FARMS) algorithm. The FARMS algorithm uses only perfect match probes and a quantile normalization procedure, providing both P-values and signal intensities . Normalised data were then further subjected to filtering for informative probe sets using the Informative/Non-informative calls (I/NI-calls) software package within R . I/NI-calls uses multiple probes for the same gene as repeated measures to quantify the signal-to-noise ratio of that specific probe set. Principal component analysis (PCA) was performed using data from all informative probe sets following all data filtering steps from the M. bovis-challenged and non-challenged control samples at the 2, 6 and 24 hour time points. The MultiExperiment Viewer version 4.7 software package was used for PCA with Euclidean distance as the distance metric.Differentially expressed genes were identified using the Linear Models for Microarray Data (LIMMA) package contained within R. Genes displaying differential expression patterns between non-challenged control and M. bovis-challenged MDM at the 2, 6 and 24 hour time points were annotated using the Affymetrix® bovine gene annotation (http://www.affymetrix.com). The Benjamini-Hochberg multiple testing correction method was applied to all differentially expressed genes to minimise the false discovery rate (FDR) and adjusted P-values for differentially expressed genes were calculated (adjusted P-value threshold 0.05).Geometric mean fold-changes in gene expression are presented in the current study following back-transformation of mean log2 fold-changes obtained from statistical analysis of microarray data. For replicate probes for single genes, the average log2 expression fold-change was used and subsequently back-transformed. The negative reciprocals of the geometric fold-changes were calculated and are presented for downregulated genes. All microarray data were MIAME compliant and have been submitted to the NCBI Gene Expression Omnibus (GEO) database with experiment series accession number GSE33309. [...] Intron-spanning primers were designed for each gene using the Primer3Plus package and commercially synthesised (Eurofins MWG Operon, Ebersberg, Germany). provides sequence information for all primer pairs used. Real time qRT-PCR reactions (20 µl final volume) were performed on 96-well plates using Fast SYBR® Green Master Mix (Applied Biosystems®, Life Technologies Corporation, Warrington, UK) on a 7500 Fast Real Time PCR System (Applied Biosystems®, Life Technologies Corporation, Warrington, UK) as per manufacturer's instructions. Real time qRT-PCR amplifications contained as template either: (a) 2 µl of the diluted conventionally-prepared cDNA/non-RT control (equivalent to 1.0 ng of total RNA); or (b) 2 µl of the diluted linearly amplified cDNA/non-RNA template control. A final concentration of 300 nM of each forward and reverse primer was included in each amplification reaction. Non-template real time qRT-PCR controls and a seven-point standard curve prepared from 1∶2 serial dilutions of pooled conventionally-prepared cDNA or linearly amplified cDNA from each M. bovis-challenged MDM sample were included on every real time qRT-PCR plate.PCR thermal cycling conditions for each amplicon comprised one cycle at 50°C for 2 minutes, one cycle at 95°C for 20 seconds, followed by 40 cycles at 95°C for 3 seconds and 60°C for 30 seconds. A dissociation step was included for all amplifications to confirm the presence of single discrete PCR products of the expected size; this was further confirmed by visualisation of the amplification products on 2% agarose gels stained with 0.5 µg/ml ethidium bromide (Invitrogen™, Life Technologies Corporation, Paisley, UK). […]

Pipeline specifications

Software tools Simpleaffy, FARMS, limma, Primer3
Applications Gene expression microarray analysis, qPCR
Organisms Mycobacterium bovis, Bos taurus, Mycoplasma bovis
Diseases Infection, Tuberculosis, Macrophage Activation Syndrome