Unknown,Transcriptomics,Genomics,Proteomics

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Hepatic transcriptional profiling of mRNA and microRNA in pigs infected with Actinobacillus pleuropneumoniae

ABSTRACT: Actinobacillus pleuropneumoniae is a gram-negative bacterium that causes porcine pleuropneumonia, which is a widespread, highly contagious, and often fatal respiratory disease in swine. In this experiment pigs were inoculated with A. pleuropneumoniae serotype 5b. Liver samples from three non-inoculated pigs and three experimental inoculated pigs were used to characterize the expression profiles of mRNA and microRNA genes using DNA microarrays and Illumina GA deep sequencing, respectively. The microarray analysis identified a large number of genes, which significantly differed in expression in infected versus non-infected animals. MicroRNAs are short single stranded RNA molecules that regulate gene expression by sequence specific binding to the 3M-BM-4 untranslated region (3M-BM-4UTR) of target mRNAs. The deep sequencing analysis determined the identity and abundance of nearly 400 microRNAs, of which a portion was found to significantly differ in expression between the infected and non-infected animals. Target genes for differentially expressed microRNAs were predicted using microCosm Targets, which is based on the miRanda algorithm. Comparison on the two gene lists showed many common genes, which may suggest a causative relationship between changes in microRNA expression and target gene expression. The expression profiles of mRNA and smallRNA in liver from three experimentally infected pigs were compared with the profiles three non-infected contol pigs.

ORGANISM(S): Sus scrofa

SUBMITTER: Jakob Hedegaard

PROVIDER: E-GEOD-32947 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The introduction of long oligonucleotide-based probes has led to many comparative studies of these two platforms in mammals, but remains to be performed for pig. Further, the characteristics of global gene expression in diverse porcine muscle tissues have not been yet been fully established. This is the first global gene expression study of a collection of nine porcine muscle tissues consisting of cardiac and various skeletal muscle types using both cDNA-based and long oligonucleotide-based microarray platforms. The expression profiles from the two platforms agree in differentiating between cardiac, skeletal red, skeletal intermediate and skeletal white muscle types by producing almost identical hierarchical expression clusters. The clusters from both platforms also reveal that gene expression profiles of the skeletal intermediate type are more similar to the red type than to the white type. Analysis of the ability to identify differentially expressed genes based on gene set analysis and GO term integration show a platform overlap of at least 80% for the cardiac-skeletal comparisons and a platform overlap of at least 58% for the skeletal red-white comparisons. Interestingly, the oligonucleotide platform was always able to identify more GO terms associated with differential expression than the cDNA platform. We further examined the skeletal red-white expression differences and found many GO biological processes that are known to be associated with these phenotypes including calcium ion transport, glycolysis, fatty acid beta-oxidation and muscle contraction. In addition, the expression of genes involved in differentiation between slow (red) and fast (white) muscle types such as MYBPC1, TNNI1, TNNT3 and ATP2A1 is highly regulated between red and white type muscles. Not previously shown to be associated with this tissue difference we found the biological process post-Golgi vesicle-mediated transport. Similar results were obtained with GO classes cellular components and molecular functions. Using the gene expression profiles from the oligonucleotide platform we presented and applied an approach for predicting alternatively spliced transcripts across cardiac and skeletal type muscles. One of the predicted transcripts from the gene named UBE2C has been shown to have six transcript variants in human, but they are not expressed in a cardiac-skeletal specific manner as we have observed here. We speculate that one of our oligonucleotide probes for this gene is able to detect only three of these variants whereas the other detects all six variants and that these variants are expressed in a cardiac-skeletal muscle specific manner. These results supports some of the advantages of using oligonucleotide-based microarray platforms for global gene expression profiling and the observed differences in gene expression among muscle tissues contribute to the understanding of the molecular processes behind porcine muscle biology. Keywords: tissue comparison, platform comparison Muscle tissue samples from heart (HEA), Vastus intermedius (VIN), Infraspinatus (ISP), Supraspinatus (SSP), Biceps femoris (BFE), Longissimus dorsi (LDO), Semimembranosus (SME), Semitendinosus (STE) and Triceps brachii (TBR). A common reference sample was constructed by combining all samples. The exact same tissue samples were used for expression profiling with cDNA microarrays and 70-mer long oligonucleotide microarrays.

Project description:The recent development within high-throughput technologies for expression profiling has allowed for parallel analysis of transcriptomes and proteomes in biological systems such as comparative analysis of transcript and protein levels of tissue regulated genes. Until now, such studies of have only included microarray or short length sequence tags for transcript profiling. Furthermore, most comparisons of transcript and protein levels have been based on absolute expression values from within the same tissue and not relative expression values based on tissue ratios. Presented here is a novel study of two porcine tissues based on integrative analysis of data from expression profiling of identical samples using cDNA microarray, 454-sequencing and iTRAQ-based proteomics. Sequence homology identified 2.541 unique transcripts that are detectable by both microarray hybridizations and 454-sequencing of 1.2 million cDNA tags. Both transcript-based technologies showed high reproducibility between sample replicates of the same tissue, but the correlation across these two technologies was modest. Thousands of genes being differentially expressed were identified with microarray. Out of the 306 differentially expressed genes, identified by 454-sequencing, 198 (65 %) were also found by microarray. The relationship between the regulation of transcript and protein levels was analyzed by integrating iTRAQ-based proteomics data. Protein expression ratios were determined for 354 genes, of which 148 could be mapped to both microarray and 454-sequencing data. A comparison of the expression ratios from the three technologies revealed that differences in transcript and protein levels across heart and muscle tissues are positively correlated. We show that the reproducibility within cDNA microarray and 454-sequencing is high, but that the agreement across these two technologies is modest. We demonstrate that the regulation of transcript and protein levels across identical tissue samples is positively correlated when the tissue expression ratios are used for comparison. The results presented are of interest in systems biology research in terms of integration and analysis of high-throughput expression data from mammalian tissues. Keywords: tissue comparison, platform comparison Tissue samples of heart (HEA) and Longissimus dorsi (LDO) were prepared by pooling from five healthy Hampshire gilts at age four to six months and division into six sub-samples, three for each tissue named HEA1, HEA2, HEA3, LDO1, LDO2 and LDO3. The exact same six tissue samples were used for subsequent expression profiling with cDNA microarray, 454-sequencing and iTRAQ-based proteomics. A reference sample for the cDNA microarray experiment and iTRAQ-based proteomics was constructed by combining the six samples. In the microarray experiment, three cDNA microarray slides were used per sample.

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Hepatic transcriptional profiling of mRNA and microRNA in pigs infected with Actinobacillus pleuropneumoniae

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