Free Circulating miRNAs Measurement in Clinical Settings: The Still Unsolved Issue of the Normalization

Abstract

Circulating molecules that are released into the circulation in response to specific stimuli are considered potential biomarkers for physiological or pathological processes. Their effective usefulness as biomarkers resides in their stability and high availability in all the biological fluids, combined with the limited invasiveness of intervention. Among the circulating molecules, miRNAs represent a novel class of biomarkers as they possess all the required characteristics such as sensitivity, predictivity, specificity, robustness, translatability, and noninvasiveness. miRNAs are small non-coding RNAs, that act as inhibitors of protein translation, and intervene in the complex network of the post-transcriptional mechanisms finely regulating gene expression. The emerging role of miRNAs as potential biomarkers for clinical applications (e.g., cancer and cardiovascular diseases diagnosis and prediction, musculoskeletal disease diagnosis and bone fracture risk prediction), however, requires the standardization of miRNA processing, from sample collection and sample storage, to RNA isolation, RNA reverse-transcription, and data analyses. Normalization is one of the most controversial issues related to quantitative Real-Time PCR data analysis since no universally accepted normalization strategies and reference genes exist, even more importantly, for circulating miRNA quantification. As it is widely demonstrated that the choice of different normalization strategies influences the results of gene expression analysis, it is important to select the most appropriate normalizers for each experimental set. This review discloses on the different strategies adopted in RT-qPCR miRNA normalization and the concerning issues to highlight on the need of a universally accepted methodology to make comparable the results produced by different studies.

Keywords: Biomarkers; Normalization; RT-qPCR; Reference genes; microRNA.

Figure 1

Biogenesis and function of miRNA. miRNA genes are transcribed from noncoding inter- or intra-genic regions of RNA transcripts. The transcription is mediated by RNA polymerase II and give rise to a long pri-miRNA that is processed by the DROSHA–DGCR8 complex to form a 60 nucleotide-long precursor miRNAs (pre-miRNAs). EXPO5 mediates the export of the pre-miRNA to the cytoplasm where it is further processed by the ribonuclease DICER that trims the pre-miRNAs to form the 18–22 nucleotide-long mature miRNAs. The guide strand is incorporated into RISC involving DICER and AGO2 enzymes to target mRNAs to cause the repression of translation and/or its degradation. This figure was produced using Servier Medical Art, available at https://smart.servier.com/.