Researchers at the University of Oxford and the Scripps Research Institute recently published an article evaluating the use of several different RT-qPCR normalization strategies when profiling miRNA gene expression in mouse serum. In their research article entitled “Assessment of RT-qPCR Normalization Strategies for Accurate Quantification of Extracellular microRNAs in Murine Serum,” Roberts et. al. compare three miRNA expression normalization strategies: 1.) normalization of miRNA expression to the average Cq (Ct) of all measured miRNA genes, 2.) Normalization of gene expression to individual miRNAs such as miR-16, miR-223, or miR-31 (all miRNA genes previously identified as normalization controls in the literature), and 3.) Normalization to a ‘spike-in’ external control miRNA gene expressed in C. elegans (cel-miR-39).
Normalization strategies have been an increasingly important topic in miRNA expression studies. This is especially true when profiling miRNA expression in serum or tissues and when quantifying miRNA genes which can have considerable variability in their endogenous expression. The authors identified a panel of 124 serum miRNA genes present in all their samples between normal, dystrophic, and dystrophic-treated mice. Their results suggest that while miR-223 and miR-16 were expressed at relatively stable levels among miRNA genes tested, miR-31 was one of the most variably expressed of the tested miRNA genes and is not suitable as a normalization control gene. The authors noted that normalization of miRNA gene expression to miR-223 or miR-16 exuded similar results to the normalizing strategy of using the average Cq value of all genes assayed together. However, the authors noted considerable variability of miR-233 and miR-16 expression levels throughout different time points and between experiment groups in this study. Roberts et. al. observed that using an external ‘spike-in’ control miRNA produced the least variability in raw Cq values. The expression ratios of all 124 miRNA genes evaluated were also higher than data normalized to an endogenous control gene, suggesting the use of an external control gene is more suitable to identify subtle changes in miRNA expression levels. The authors also provide evidence to suggest that an external ‘spike-in’ control not only produces the least amount of variability, but this strategy is effective in accounting for miRNA expression bias resulting from RNA extraction efficiencies and changes in global miRNA expression levels dependent on experimental conditions.
This research article highlights and contributes to an important discussion concerning normalization strategies when quantifying miRNA expression in serum and tissue samples. The authors provide important evidence supporting the use of an external ‘spike-in’ control for miRNA expression normalization in a dystrophic mouse model system and argue that the use of a ‘spike-in’ will allow researchers to identify accurate and even subtle changes in miRNA gene expression throughout different experimental models and/or disease states.