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Open Access Highly Accessed Research article

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro

Nicolas C Nalpas1, Stephen DE Park1, David A Magee1, Maria Taraktsoglou1, John A Browne1, Kevin M Conlon2, Kévin Rue-Albrecht1, Kate E Killick1, Karsten Hokamp3, Amanda J Lohan4, Brendan J Loftus4, Eamonn Gormley5, Stephen V Gordon24 and David E MacHugh14*

Author affiliations

1 Animal Genomics Laboratory, UCD School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Dublin 4, Ireland

2 UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Dublin 4, Ireland

3 Smurfit Institute of Genetics, Trinity College Dublin, Trinity College, Dublin, Ireland

4 UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Dublin 4, Ireland

5 Tuberculosis Diagnostics and Immunology Research Centre, UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Dublin 4, Ireland

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Citation and License

BMC Genomics 2013, 14:230  doi:10.1186/1471-2164-14-230

Published: 8 April 2013

Abstract

Background

Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA.

Results

A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology.

Conclusions

This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA.

Keywords:
Bovine tuberculosis; Immune response; Microarray; Mycobacterium bovis; Natural antisense transcript; RNA-sequencing; Transcriptome