Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 Page 5 of 11 Figure 3 Normalisation of miR-Q arrays using a set of 5 reference snRNAs. Triplicate detection of reference snRNAs (SNORD44, SNORD47, SNORD48, SNORD52 and RNU6) in human primary macrophages of three independent donors that were infected (samples) with Mycobacterium avium hominissuis or remained non-infected (calibrators) is shown Figure 3A. The measurement revealed minor Cq variation in samples and calibrators while all negative controls (NC) remained negative across 40 cycles. Light colours represent the infected samples and dark colours the non-infected calibrators. Figure 3B shows melting curves proving the specificity of all amplicons. Dissociation analysis was performed by initial denaturation at 95°C for 1 min, amplicon hybridisation at 60°C for 2 min and melting by ramping from 60°C to 95°C at 2°C/ min and acquiring the fluorescence signal. Figure 3C evaluates the stability of the chosen reference snRNAs among donors as well as samples (infected) and calibrators (non-infected) as demonstrated by the coefficient of determination r2 = 0.9788 (P < 0.0001). this purpose, fold differences were calculated from ΔCq between samples and calibrators. The calculated M values for the entire set were below 0.5 (MSNORD44 = 0.37; MSNORD47 = 0.41; MSNORD48 = 0.44; MSNORD52 = 0.3; MRNU6 = 0.31) pointing also to high stability of presented reference genes and underlining the correlation analysis. This set of 5 reference snRNAs was also highly stable in other macrophages as well as intestinal samples (data not shown). For comparative quantification analysis based on the ΔΔCq algorithm we emphasise the evaluation of the stability of reference snRNAs in the studied samples as described above. The geometric mean of the most stable candidates among the set of 5 can then be chosen for nor- malisation of miRNA expression values (ΔCq = Cq miRoI - Cq norm). We encourage the use of at least 3 reference snRNAs for normalisation of miRNA expression. miR-Q arrays are highly reproducible and accurate A measure for high reproducibility of RT-qPCR arrays is the evaluation of inter-assay variance. We assessed the reproducibility of miR-Q arrays by performing RT reac- tion from 6 independently differentiated THP-1 passages that were measured on 6 independent miR-Q array runs. For this purpose, THP-1 monocytes were differentiated into macrophages and after 28 h samples were taken and RT reactions were performed in parallel. This introduced experiment outlines a dual challenge to determine the reproducibility since independent biological samples were employed for determination of the inter-assay var- iance. Figure 4A shows the inter-assay distribution of normalised values (ΔCq = Cq miRoI - Cq norm) of 6 repli- cates each representing the mean of triplicate intra-assay measurement. As mentioned above the value Cq norm was calculated as the geometric mean of the set of 5 stable reference snRNAs. In consistence with the data shown in Figure 2B, 10 miRNAs were not expressed in differen- tiated THP-1, or were below the detection limit. As shown by the box plots in Figure 4, the other 16 showed highly constant expression among the 6 biological repli- cates and independent runs, respectively. This particu- larly points out the high reproducibility of miR-Q arrays but also the studied biological system. For comparative analyses we highly recommend performance of simulta- neous RT reactions of all samples included in a study using a thermal cycler. The preparation of master mixes also minimises inter-assay variance. Additionally, we have performed spike-in experiments to check for the accuracy of the miR-Q assays. For this pur- pose, we have selected three miRNAs (let-7e, miR-143 and miR-145) that were proven to be absent in differentiated THP-1 (Figure 4A). RT reactions as performed for the vali- dation of reproducibility were spiked with synthetic let-7e, miR-143 and miR-145 to give a final concentration in RT- qPCRs at 3.3, 33 and 330 pM, respectively. According to