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).