Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 R E S E A R C H A R T I C L E Open Access Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes Soroush Sharbati*, Jutta Sharbati, Lena Hoeke, Marc Bohmer and Ralf Einspanier Abstract Background: Small 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. Results: We 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. Conclusions: The 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-b 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. Keywords: microRNA, RT-qPCR, array, normalisation, isomiR, Salmonella, macrophage Background Metazoan regulation of gene expression relies on an interwoven network of e.g. DNA methylation, transcrip- tion factors, mRNA degradation or translational control. Recent research has shown that translational regulation as well as mRNA degradation is controlled by RNA inter- ference (RNAi). The main class of intrinsic small regulat- ing RNAs concerting these effects in eukaryotes is constituted of microRNAs (miRNAs). Mature miRNAs (about 20 nt length) derive from a hairpin. The active strand is loaded to an Argonaute family protein to form the miRNA induced silencing complex (miRISC), which * Correspondence: sharbati@zedat.fu-berlin.de Institute of Veterinary Biochemistry, Freie Universitaet Berlin, Berlin, Germany recognises the target site within a 3’ UTR. Animal miR- ISC was originally thought to repress target translation rather than mRNA degradation. However, recent data suggest that mRNA degradation may be the predominant mode of miRNA mediated regulation of gene expression [1]. Accordingly, various studies have shown negatively correlated expression of miRNAs and their targets [2,3]. Based on these observations, in silico tools such as MAGIA [4] were developed to link target prediction to the expression analysis of miRNAs and their target mRNAs. Connecting negatively correlated miRNA and target mRNA expression with target prediction allows for the identification of aberrations in miRNA mediated reg- ulation among various disease related pathways. The role © 2012 Sharbati et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.