A novel and universal method for microRNA RT-qPCR data normalization

Abstract

Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.

Figure 1

geNorm expression stability plot. Expression stability of 13 different small RNA controls and the mean expression value in the T-cell acute lymphoblastic leukaemia sample set. The mean expression value shows the highest expression stability across all 49 samples analyzed.

Figure 2

Cumulative distribution of miRNA coefficient of variation (CV) values. The cumulative distribution of miRNA CV values in the neuroblastoma sample set when no normalization is applied (blue), stable RNA control (RNU24, RNU44, RNU58A and RNU6B) normalization is applied (red), mean expression value normalization is applied (green) or normalization with miRNAs/small RNA controls resembling the mean expression value (Z30, RNU24, miR-361, miR-331 and miR-423) is applied (purple).

Figure 3

Differential miR-17-92 expression in neuroblastoma tumor samples. Average fold change expression difference of eight different miRNAs residing within the miR-17-92 cluster in MYCN amplified neuroblastoma samples compared to MYCN single copy neuroblastoma samples. Fold changes were calculated upon stable small RNA control (RNU24, RNU44, RNU58A and RNU6B) normalization (dark grey), mean expression value normalization (light grey) and normalization with miRNAs that resemble the mean expression value (miR-425, miR-191 and miR-125a; medium grey.

Figure 4

Cumulative distribution of Spearman's Rank rho values. The cumulative distribution of the Spearman's Rank rho values for each individual miRNA in the neuroblastoma sample set. The rho-values represent the degree of correlation between the miRNA expression profile upon mean expression value normalization or normalization with miRNAs resembling the mean expression value.