Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 we suggest designing mismatched base pairing at the 3’ OH of the ‘miRNA’-rev oligonucleotides. However, the respective target recognising part of the reverse primer has to be as long as possible to ensure proper hybridisa- tion at 60°C. Nevertheless, while designing a new miR-Q assay, one has to take into account that an overlap of more than 3 nucleotides between RT6-’miRNA’ and ‘miRNA’-rev should be avoided because it results in high background of assays. Recent deep sequencing efforts of small RNAs have shown that many mature miRNAs are either trimmed alternatively resulting in 5’ and 3’ variations or additional 3’ non-template adenosines or uridines are transferred by nucleotidyl transferases post-transcriptionally [21]. These variants of the canonical miRNA are often referred to as ‘isomiRs’. We were interested how far the miR-Q approach is biased toward detecting only the canonical mature miRNA without any additions or if it is possible to quantify the whole population of a given miRNA including isomiRs. Therefore, we performed additional experiments evaluating the bias of our approach by non-template ade- nosines or uridines present at the 3’ end of non-canonical isomiRs based on miR-24 variants. Recently, this miRNA was shown to possess additional 3’ adenosine or uridine modifications [22]. We performed comparative and paral- leled spike-in experiments using synthetic canonical miR- 24 as well as the A or U isomiRs, respectively. As it is shown in Figure 5C, the design of the miR-Q approach allowed the detection of isomiRs regardless of an addi- tional adenosine or uridine. All variants were detected over the same linear range proving the coverage of the entire population. We outlined a correlation matrix by computing the Pearson correlation coefficient (r) for every pair of data sets. The analysis revealed high linearity between all data sets possessing r < 0.9969 and two-tailed P values < 0.0001 (Figure 5D). The miR-Q approach pro- vides an advantage over other detection chemistries based e.g. on hairpin reverse primers allowing to capture the entire expression of a given miRNA including the canoni- cal molecule as well as isomiRs. Salmonella infection of human macrophages results in specific miRNA response proven by miR-Q arrays The developed protocol was employed to investigate miRNA regulation in monocyte differentiation after PMA treatment as well as Salmonella infection. We studied miRNA regulation in three groups using the monocytic THP-1 as a differentiation model. The first group con- sisted of monocytic THP-1 cells that were stimulated with viable Salmonella. The second group represented macrophages derived from PMA treated THP-1, while the third was composed of differentiated macrophages that were infected with viable Salmonella. All groups were compared to the untreated controls performing Page 8 of 11 triplicate measurements of triplicate biological samples. The correlation coefficients between the entire set of reference snRNAs of samples and controls ranged from 0.9884 to 0.9997 pointing out the high linearity and stabi- lity of the chosen reference snRNAs. All three treatments resulted in up-regulation of miR-146a/b from 5 to more than 10 fold (Figure 6), while only PMA treatment increased the miR-155 expression 3 fold. These results are consistent with the study of Taganov and colleagues, who reported comparable up-regulation of these miRNAs [23]. It furthermore underlines the accuracy of the pre- sented protocol. Interestingly, miR-222, miR-21 and miR-24 were up-regulated after differentiation while 2 h stimulation of monocytes with viable Salmonella did not affect their expression (Figure 6). All three miRNAs were recently reported to play roles in monocyte differentia- tion into macrophages as well as dendritic cells [24,25]. While miR-23b possessed averaged 1.4 fold decreased expression 2 h post Salmonella infection of monocytic THP-1, PMA treatment increased its expression more than 2 fold in both groups. This is the first study report- ing miR-23b dysregulation in monocyte stimulation and differentiation. Down-regulation of miR-23b was related to activation of the TGF-b1/Smad3 signalling pathway in hepatocytes [26]. TGF-b on the other hand is known to be a monocyte activator triggering cytokine production and mediating host defence [27]. Therefore, we hypothe- sise a miR-23b dependent TGF-b regulation in mono- cytes encountering pathogens or pathogen-associated molecular patterns (PAMPs) leading to host defence mechanisms. We found miR-27a to be only up-regulated in differentiated and infected THP-1. While miR-27a/b were recently reported to have antiviral effects in murine primary macrophages [28], this is the first study reporting increased expression of monocytic miR-27a upon Salmo- nella interaction. Accordingly, differentiation of THP-1 into macrophages led to more than 3 fold increased expression of miR-29a and miR-29c. The miR-29 family turns out to be a key regulator in host immune response to bacterial pathogens as it was recently shown to control the immune response to intracellular bacteria by target- ing interferon-g and to regulate apoptosis of primary human macrophages in mycobacterial infections [6,8]. Conclusions The presented approach is the first custom and non- commercial protocol for RT-qPCR arrays based on a miRNA specific RT-qPCR called miR-Q. The developed miR-Q arrays enable reliable and feasible quantification of small non-coding RNAs as exemplified by miRNA expression analysis during monocyte differentiation and infection. On one hand, well known miRNA dysregula- tion verified the accuracy of the presented method. On the other hand, several dysregulated miRNAs were newly