Measurements Methods for the Development of MicroRNA-Based Tests for Cancer Diagnosis

Abstract

Studies investigating microRNAs as potential biomarkers for cancer, immune-related diseases, or cardiac pathogenic diseases, among others, have exponentially increased in the last years. In particular, altered expression of specific miRNAs correlates with the occurrence of several diseases, making these molecules potential molecular tools for non-invasive diagnosis, prognosis, and response to therapy. Nonetheless, microRNAs are not in clinical use yet, due to inconsistencies in the literature regarding the specific miRNAs identified as biomarkers for a specific disease, which in turn can be attributed to several reasons, including lack of assay standardization and reproducibility. Technological limitations in circulating microRNAs measurement have been, to date, the biggest challenge for using these molecules in clinical settings. In this review we will discuss pre-analytical, analytical, and post-analytical challenges to address the potential technical biases and patient-related parameters that can have an influence and should be improved to translate miRNA biomarkers to the clinical stage. Moreover, we will describe the currently available methods for circulating miRNA expression profiling and measurement, underlining their advantages and potential pitfalls.

Diagnostic and prognostic potential of eight whole blood microRNAs for equine sarcoid disease

“…RT-qPCR was employed for miRNA analysis in this study, since it shows excellent sensitivity, specificity, and reproducibility, requires only widely available standard equipment, and is thus more suitable for routine use than NGS [ 30 ]. Relative miRNA quantification methods are most commonly applied when using RT-qPCR [ 46 ]. The two normalization methods used in this study demonstrated overall good agreement.…”

“…Regarding data normalization, the choice of appropriate reference genes is of particular importance in order to avoid bias in miRNA quantification. There is, however, neither a universally valid endogenous control nor a consensus on the most appropriate references miRNAs in biofluids [ 46 , 47 ]. We based our choice on miRNA expression data from the exploratory NGS study, which perfectly reflected the nature of the samples included in the current validation study [ 28 , 46 ].…”

“…There is, however, neither a universally valid endogenous control nor a consensus on the most appropriate references miRNAs in biofluids [ 46 , 47 ]. We based our choice on miRNA expression data from the exploratory NGS study, which perfectly reflected the nature of the samples included in the current validation study [ 28 , 46 ]. Eca-miR-30d was the only miRNA in our dataset that qualified as endogenous control.…”

“…However, due to the significant influence of biological variation on miRNA expression, no universally valid miRNA biomarker currently qualifies for clinical applications. Additionally, miRNAs are not yet seeing clinical use as biomarker in human medicine, due to lack of assay standardization and reproducibility of results among studies [ 46 ]. Nevertheless, it is warranted to further explore the proposed diagnostic and prognostic miRNA biomarkers for ES disease in larger study cohorts.…”

The Role of miR-23b in Cancer and Autoimmune Disease

“…miRNAs have been involved in many cancers and neurodegenerative diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease [ 37 , 38 ]. Overall, miRNAs play an essential part in the occurrence and development of diseases [ 39 ].…”