Fig 1.
Analysis pipeline for comparison of missing data and data normalization handling strategies impact on identification of stable endogenous miRNAs controls and survival analysis.
Only miRNAs without missing values (complete_cases) were included to assess their stability with five algorithms: the comparative delta-Ct method (deltaCt), BestKeeper, NormFinder, geNorm, and RefFinder. Our findings were further validated in external cohorts. Single or multiple imputation methods were applied on miRNAs with missing value ratio lower than 20%. Three normalization strategies were used individually on different input datasets. Finally, survival analysis by Cox regression was performed on fifteen differently processed datasets originated from the same RT-qPCR raw dataset.
Table 1.
Stable endogenous control and biomarker candidates investigated in the study.
Table 2.
The expression values of miRNAs and controls presented as their mean (mean Cp), standard deviation (sd) and ratio of missing values (ratio NA).
Table 3.
Stability values and rankings for five algorithms used to identify best stable endogenous controls.
The stability values are not directly comparable between different algorithms.
Table 4.
Characteristics of external cohorts included in the validation study.
Table 5.
Comparison of stability rankings for shared miRNAs between our cohort and two external datasets: GSE81873 and GSE43867.
Table 6.
Comparison of stability rankings for selected miRNAs in different subsets of two external datasets: GSE81873 and GSE43867.
Table 7.
Coefficient of variation analysis on individual miRNAs that presented missing Cp values and were imputed with four methods (missF, VIM, na_max_one, na_35).
CoV, CoV_imp_missF, CoV_imp_VIM, CoV_imp_max_one, and CoV_imp_na_35 –coefficient of variation for particular RNA in a raw dataset, in the missF imputed dataset, in the VIM imputed dataset, in max_one imputed dataset and na_35 imputed dataset, respectively.
Table 8.
Univariate Cox analyses to identify the candidate predictors for OS.
miRNAs with P-value below 0.05 are presented.