Validation of suitable reference genes for quantitative expression analysis by qPCR in bovine terminal ileum and ileocecal valve.

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The aim of this study was to identify a set of reference genes suitable for gene expression analysis in the distal portion of small intestine and ileocecal valve in cattle. These sites of intestine are of interest in veterinary science as they are the main sites of inflammation caused by Mycobacterium avium subsp. paratuberculosis, agent of paratuberculosis. We employed ten PCR assays for commonly used reference genes belonging to various functional classes and then determined their expression stability. The most stable genes were RPL13A and HMBS, followed by TFRC and B-ACT. NormFinder analysis provided similar results with B-ACT as the best reference gene, followed by RLP13A and TFRC. This validated gene panel may be useful for studies on paratuberculosis aiming to identify genes differentially expressed by qRT-PCR.

Polymorphisms associated to bovine paratuberculosis: investigation of their role in DNA-protein interactions and transcriptional regulation.

Previous studies led to identify SNPs in putative regulatory regions of the SLC11A1 and CARD15 genes with association to paratuberculosis in cattle. Aim of this study was to investigate the role of these mutations at the regulatory level by DNA-protein interaction analyses and transcriptome comparison between wild-type and mutated animals. Gene regions carrying the SNPs of interest were analysed by bioinformatic tools to predict allele-dependent binding sites for transcription factors (TFBS). Putative TFBS were in vitro explored by Electrophoretic Mobility Shift Assays (EMSA). EMSA did not show specific gel shifts for any allele indicating that these SNPs may eventually influence gene transcription without altering TFBS. Whole transcriptome expression analysis was performed on intestinal tissues of wild-type and mutated cattle by RNA-Seq. Differential regulation of five genes involved in innate immune system was detected. Specifically, ULBP3 was down-regulated, while S100A8, S100A12, LOC510860, and IFI27 were up-regulated. In previous studies, ULBP3, S100A8, and S100A12 resulted differentially expressed in cattle affected by paratuberculosis, suggesting a possible implication in the pathogen response. Further investigations are needed to elucidate the functional role of these SNPs and to understand the gene network involved in the interactions between non-coding SNPs and other genome regions.