Computational protocol: Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients

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Protocol publication

[…] Normalization analysis was performed using different online available normalization tools. The first one is NormFinder v0.953 (, which works with an algorithm for linear data to determine the most stable normalization candidate gene []. The tool calculates a stability value (SV) for every single candidate. The lower the stability value, the more stable is the expression of the corresponding candidate gene. The data output comes with the stability values for all candidates together with standard deviations (SD) and announces the most stable candidate gene. Splitting data into study groups, results in additional intra- and intergroup variation and determination of the best endogenous normalizer combination. The second tool used in this study is the online-based tool RefFinder [] ( It comprises four different commonly used normalization tools, namely BestKeeper [], comparative DeltaCt [,], NormFinder [] and GeNorm [], working with different algorithms to evaluate the most stably expressed gene or gene pair of a specific sample set. [...] All statistical analyses were performed with IBM SPSS Statistics 22.0 (SPSS Inc., Chicago, IL, USA). Data was tested for normality by Kolmogorov-Smirnov test. Differences between two groups were tested with Mann-Whitney-U-test. The two-tailed tests with p-values ≤ 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools NormFinder, BestKeeper, SPSS
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Cardiovascular Diseases, Renal Insufficiency, Chronic