Application of Next Generation Sequencing (RNA-seq and miRNA-seq) to study the molecular signature of Aluminium hydroxide adjuvant in ovine peripheral blood mononuclear cells (PBMCs) [miRNA-Seq]

We report the application of miRNA sequencing technology for high-throughput profiling of the aluminium hydroxide adjuvant mechanism of action in an in vivo experiment on sheep. We mapped about 13 million sequence reads per sample to the sheep genome (build Oar3.1) and identified 95 miRNAs in the PBMCs of sheep treated with commercial vaccines or with the adjuvant alone. A total of 3, 6 and 1 differentialy expressed miRNAs were identified in the vaccine tf VS vaccine t0, adjuvant tf VS adjuvant t0 and adjuvant tf VS vaccine tf comparisons, respectively. Furthermore, miRNA target prediction was performed and it was integrated with previous RNA-seq data in the same animals to predict reliable miRNA-mRNA interactions. Previously reported miRNAs related to alterations caused by aluminium adjuvant were found differentially expressed, namely: miR-19b and miR-125b. Within the negatively correlated targets of the differentially expressed miRNAs there are factors that are clearly related with the response to stimulus, RNA binding and cellular response to DNA damage. Taken together, our analysis provides characterization of alterations in the miRNAome caused by the aluminium adjuvant and identifies some targets of the differentially expressed miRNAs which have been previously linked to the immune system. The overrepresentation of genes related to DNA damage stimulus and DNA repair in both groups may be due to miRNA-mediated regulation. Overall design: PBMCs miRNA profiles of six age-matched Rasa sheep before and after inoculation (t0 and tf) of nine different commercial vaccines based of aluminium hydroxide (the vaccine group composed of 3 animals) or with the aluminium adjuvant alone in an equivalent dose (the adjuvant group composed of 3 animals) were generated by deep sequencing on an Illumina HiSeq2500 sequencer. In total, the study is composed of three biological replicates per group and twelve libraries were sequenced.