Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

Abstract

BackgroundSmall interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes.ResultsWe have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively.ConclusionsThe provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-β signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages

“…The role of miRNAs has been documented in the process of hematopoiesis, differentiation, survival, and function of immune cells, including monocytes and macrophages (23,54). Transfection of miR-29b, miR-125a-5p, or miR-155 mimics in THP-1 cells altered macrophage polarization to M1 macrophages (23).…”

“…The role of miRNAs has been documented in the process of hematopoiesis, differentiation, survival, and function of immune cells, including monocytes and macrophages (23,54). Transfection of miR-29b, miR-125a-5p, or miR-155 mimics in THP-1 cells altered macrophage polarization to M1 macrophages (23). Experiments with human macrophages have identified unique miRNAs associated with polarized macrophage phenotypes such as miR-155 in M1 macrophages and miR-27a and miR-132 expression in M2 macrophages (20).…”

“…Increasing evidence suggests that miRNAs, a class of non-coding RNAs, play an important role in macrophage polarization (7, 20 -22). We and others have shown that miRNAs can regulate cellular differentiation and functions in immune cells (21,23,24). miRNAs not only function within the cells of origin but can also be packaged into EVs and released into the extracellular environment, where they are transferred in an active form to other cells and exert functional effects (16,(25)(26)(27)(28).…”

Current and Emerging Innovations for Detection of Food-Borne Salmonella

“…The "real-time" aspect of real-time PCR, also referred to as qPCR, technology is linked to its ability to label and cumulatively quantify the generated PCR products at each cycle throughout the ongoing ampliication process. The qPCR has been widely used to quantify Salmonella [54][55][56]. Daum screened nine foodstufs associated with a Salmonella outbreak in Texas using qPCR.…”

MicroRNAs in Human Microbial Infections and Disease Outcomes

“…It has also been described that the expression of miR-146a is upregulated by this pathogen in gastric epithelial cells and mucosa tissues in a NF-κB-dependent manner. In addition, upregulation of these two important miRNAs has been observed likewise in response to the intracellular infection caused by Salmonella [11,[20][21][22]. Schulte et al [21] demonstrated that members of the let-7 miRNA family were downregulated by Salmonella in macrophages and epithelial cells.…”