Sharbati et al. BMC Genomics 2012, 13:23 http://www.biomedcentral.com/1471-2164/13/23 Page 4 of 11 Figure 1 Efficiency of miR-Q assays. The Tukey box plots show the efficiencies of 39 miRNA- as well as 5 reference snRNA-specific miR-Q assays. A paired t test proved that there was no statistically significant difference between the efficiencies of miRNA- and reference snRNA-specific miR-Q assays (P = 0.6836). arrays. Figure 2B shows the triplicate Cq values using the layout shown in Figure 2A. Except for 10 miRNAs (let-7 family, miR-106a, miR-141, miR-143, miR-145, miR-27a and miR-29b) all other miRNAs and reference snRNAs were detected in differentiated THP-1 showing minor Cq variation in triplicate measurements and demon- strating strong repeatability of miR-Q arrays (Figure 2B). Commercially available RT-qPCR arrays often employ sin- gle measurement for individual miRNAs, so that the lack of intra-array replicates weakens the repeatability of assessed data. Furthermore, there is no opportunity to eval- uate if an undetermined Cq is based on target absence or failed reaction. For generation of highly reliable results we encourage intra-array triplicate measurements of each target. A set of 5 snRNAs allows for accurate normalisation of miRNA expression Accurate normalisation of gene expression measures is a crucial process that controls for bias based on extraction yield, RT yield and amplification efficiency. This step is the basic prerequisite for comparing gene expression values among different samples [16]. For normalisation of miR-Q array data, we have designed assays for a set of 5 snRNAs: SNORD44, SNORD47, SNORD48, SNORD52 and RNU6. We tested for applicability of these reference snRNAs using human primary macrophages from three independent donors that were infected with Mycobacter- ium avium hominissuis or remained non-infected described in a previous study [8]. As shown in Figure 3A, all reference snRNAs were detected among the three donors showing minor Cq variation in samples (infected) and calibrators (non-infected) while all negative controls (NC) remained negative across 40 cycles. Melting curve analysis proved all amplicons to be specific (Figure 3B) and RT negative controls revealed the absence of contami- nating genomic DNA (data not shown). For evaluating the stability of the chosen reference snRNAs among donors as well as samples and calibrators, we have plotted Cq values of samples versus calibrators (Figure 3C). The correlation analysis revealed high linearity between Cq sample and Cq calibrator possessing a coefficient of determination close to 1 (r2 = 0.9788 and P < 0.0001) and pointing to excellent sta- bility of all reference snRNA. This result was also verified by calculating the geNorm stability measure M [20]. For Figure 2 Arrayed quantification of miRNAs using a 96 well format. A miR-Q array layout is exemplified in Figure 2A using a 96-well format. This layout provides triplicate quantification of 26 miRNAs, 5 reference snRNAs and negative controls (NC). Figure 2B shows the Cq variations of triplicate measurements using 1 μg total RNA from differentiated THP-1. Rhombi indicate the individual measurements of each assay, while their mean is given as black lines. Black rhombi represent assays possessing specific products and blue rhombi show unspecific detection. The SYBR Green background is given as red dotted line.