Open Access Research article

Profiling microRNAs in lung tissue from pigs infected with Actinobacillus pleuropneumoniae

Agnieszka Podolska12, Christian Anthon13, Mads Bak34, Niels Tommerup34, Kerstin Skovgaard5, Peter MH Heegaard5, Jan Gorodkin13, Susanna Cirera1* and Merete Fredholm13

Author Affiliations

1 Department of Veterinary Clinical and Animal Sciences, Section of Anatomy, Cell Biology, Genetics and Bioinformatics, University of Copenhagen, Faculty of Health and Medical Sciences, Copenhagen, Denmark

2 Biotech Research & Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark

3 Center for non-coding RNA in Technology and Health, University of Copenhagen, Copenhagen, Denmark

4 Department of Cellular and Molecular Medicine, Panum Institute, Copenhagen, Denmark

5 Innate Immunology Group, National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark

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BMC Genomics 2012, 13:459  doi:10.1186/1471-2164-13-459

Published: 6 September 2012

Additional files

Additional file 1:

Profiles of read clusters for novel miRNAs from miR-d1 to miR-d6.

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Additional file 2:

Profiles of read clusters for novel miRNAs from miR-d7 to miR-d12.

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Additional file 3:

The known miRNAs of the miRDeep2 pipeline missed by the unique mappings from Novoalign. 24 of these miRNAs are found twice in the genome, indicating assembly errors. Raw reads are un-normalized and according to the Bowtie alignment produced by miRDeep.

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Additional file 4:

Expression of the all the detected miRNAs. Columns 6 and 7 contain the raw reads in the two samples. Columns 8 and 9 are the read counts normalized by the TMM method. Column 10 is log2 of the fold change based on the normalized read counts. Column 11 is the p-values for the relative expression based on the exact test from the edgeR package using a dispersion-factor of 0.1. (see Methods for details).

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Additional file 5:

Top 20 snoRNAs in the necrotic tissue. Infernal e-values are given for the annotation.

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Additional file 6:

Top 20 snoRNAs in the unaffected tissue. Infernal e-values are given for the annotation.

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Additional file 7:

Bar diagram showing RT-qPCR results of expression of 13 selected unique miRNAs and one snoRNA. miR-d5 represents a novel unannotated microRNA. All eight sample groups included.

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Additional file 8:

Statistical analysis of qPCR results:One way ANOVA performed on four groups: control, visually unaffected, demarcation zone, necrotic.

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Additional file 9:

Targets predicted for 12 select miRNAs and 91 select proteins. Only targets predicted by two or more of the four target prediction methods (see Methods for details).

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Additional file 10:

Conservation of miRNAs found in this article. In column 2–5 the conservation is based on full genomic pairwise alignments of pig and 17 other mammalian genomes (bosTau4, turTru1, equCab2, felCat3, canFam2, eriEur1, hg19, tarSyr1, mm9, rn4, oryCun2, loxAfr3, echTel1, dasNov2, choHof1, monDom5, ornAna1). The miRNA pig coordinates are required to transfer to the target organism, and the coordinates in the target organism is required to transfer back to the same position in pig to avoid paralogous alignments. The pig sequence is then aligned to the target sequence and a minimum align length of 50 and an identity of at least 50% is required for the miRNA to be counted in the third column. In columns 2 and 3 the pairwise identity between the pig sequence and cow or human is given, and in column 4 the number of organisms where the miRNA is found is given and finally in column 5 the mean pairwise identity is given. Columns 6–9 are analogous to column 2–5, except the identification of the miRNAs in the target organism are now identified by a BLAST of mirBase version 18 against the target genome. The pig and target sequence are then aligned in the same way as before.

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Additional file 11:

Targets predicted with TargetScan for mir-d5 and mir-d11 for which the set of conserved target sites gave few results. The targets are predicted for 3’ UTRs from cow since the pig ones are not in the dataset for TargetScan (see Methods for details).

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Additional file 12:

Table listing values describing the quantity and integrity of all RNA samples.

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Additional file 13:

Table listing RT-qPCR primer sequences for each assayed miRNA and snoRNA. * indicates reference genes.

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Additional file 14:

Statistical analysis of qPCR results. One way ANOVA performed on eight groups: trachea, nose, F50, F100, control, visually unaffected, demarcation zone, necrotic.

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