Identification of mouse serum miRNA endogenous references by global gene expression profiles

Abstract

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.

Figure 1. Global expression of serum miRNAs in different mouse strains.

Serum RNA was isolated from different aged (w = weeks) mouse strains (n = 20), with each time point duplicated. The globally normalized ΔC_T values are represented in the heat map with miRNA species ordered by hierarchical clustering. miRNA expression was analyzed by TaqMan® miRNA arrays and normalized by StatMiner® global normalization.

Figure 2. Five serum miRNAs as endogenous references stably expressed across different mouse strains.

−ΔC_T values of miR-146a, miR-16, miR-195, miR-30e, and miR-744 show stability across all samples (w = weeks). The −ΔC_T average of all five miRNAs is also displayed. Each gene is expressed across all samples, has an ANOVA p>0.3, an SD<1 and all pair-wise |ΔΔC_T|<0.5.

Figure 3. MammU6 is differentially expressed in the different mouse strains.

Bar plots of the average MammU6 expression for each mouse strain are given with standard error bars. An ANOVA comparison detects a significant difference in the means, p-value of 0.049, an LSD p-value of 0.023 (B6 vs. NOD), and common SD of 2.4.