Computational protocol: Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation

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Protocol publication

[…] The distributions of the qPCR quantification cycle (Cq) values of the five reference genes over the whole sample set are shown in , while the descriptive statistics given by BestKeeper are reported in . Reference genes showed different expression values and variability levels. 5S showed the largest expression (mean Cq = 18.97), while SNORD44 was the least expressed (mean Cq = 26.77). In terms of variability, miR-16 and SNORD48 displayed the lowest standard deviation (SD) values of 0.70 and 0.72, respectively. Conversely, 5S and U6 showed variability levels beyond the limit of acceptance for reliable normalizers (>1). Correlation analysis was performed between each pair of reference genes and between each reference gene and the BestKeeper Index (BKI). In particular, BKI (5) was calculated including all reference genes, while BKI (3) was obtained excluding the two genes with unacceptable variability (U6 and 5S). The analysis, shown in and in , pointed out the existence of significant correlations for SNORD48, SNORD44 and 5S when compared to one another, and for U6 when compared to 5S, while miR-16 did not correlate with any other reference gene. Consistently, all reference genes but miR-16 correlated with BKI (5). When compared with BKI (3), SNORD48 and SNORD44 showed the highest correlations (r > 0.8 and p < 0.001), while miR-16 displayed a lower, non significant correlation value (r = 0.45, p = 0.06). Considering both variability and correlation information, SNORD48 showed the best performance, combining a low SD value with high correlation values. [...] The stability analysis performed by NormFinder is displayed in in terms of stability index ρ (upper panel) and inter-group (box) and intra-group (whiskers) variations (lower panel). The worst overall performance was shown by U6 (ρ = 0.54), which displayed large values of both inter-group (−0.37) and intra-group variability (1.12). Overall performance was low also for 5S and miR-16 (ρ = 0.42 and 0.46), mainly due to high intra-group variability values (0.67 and 1.36). Best overall performance was observed for SNORD48 (ρ = 0.34), which displayed intermediate inter-group variation (0.25) and the smallest intra-group variability (0.06).The average stability index ρA, calculated for each pair of reference genes, showed that stability could be improved by using combination of reference genes with intergroup variability of opposite sign. In particular, combination of either of SNORD48 and SNORD44 with U6 led to an average stability index of ρA = 0.27, while combination with 5S led to a stability index of ρA = 0.29.The comprehensive ranking of gene stability obtained by combining the four analyses ( and ) assessed SNORD48 as the most stable gene (best performance according to all analyses), followed by SNORD44, 5S, and miR-16. U6 was the least stable gene, displaying the worst performance according to all analyses. […]

Pipeline specifications

Software tools BestKeeper, NormFinder
Application qPCR
Organisms Homo sapiens
Diseases Cardiovascular Diseases, Heart Diseases