Seeking a standardized normalization method for the quantification of microRNA expression

Assessment of salivary microRNA by RT-qPCR: Challenges in data interpretation for clinical diagnosis

“…Salivary miRNA biomarkers have become recently relevant to the medical diagnostic community due to their easiness in sample collection and their expression signatures in diverse diseases [4,6,7]. However, the lack of standardized approaches in experimental design, method of analysis and data interpretation coupled with inter-individual variability has led to the generation of considerable study-dependent and controversial data [37,39]. We evaluated the capacity to detect salivary miRNAs of a commercially available kit, which is widely used in both research and clinical studies [16,19,40].…”

“…The majority of the miRNA studies have been standardized by microarray or Next Generation of Sequencing (NGS) methods followed by subsequent validation using various customized RT-qPCR approaches [31][32][33][34][35][36]. Notwithstanding the practicality, rapidity and cost effectiveness [8] of the RT-qPCR technique, its application in the quantification of miRNAs faces methodological inconsistencies in both detection and data normalization [37][38], making data interpretation controversial [39] and study dependent [18]. These unsettled topics are the main issues that restrains the use of miRNAs in current clinical practice.…”

“…19,20,21,22 In general, these techniques mainly focus on the conjugation of a synthetic oligomer to the miRNA target, which enables the downstream amplification by qPCR at the cost of introducing partial crosstalk due to the shared synthetic oligomer. Moreover, data normalization in miRNA studies remains unclear, 4,5 where miRNA signatures differ depending on the study 23 and on the method of normalization. 24 Altogether, this makes the exploitation of salivary miRNA biomarkers for clinical diagnostics highly requested, and hence requiring highly robust yet specific quantification assays.…”

Identification and Application of Emerging Biomarkers in Treatment of Non-Small-Cell Lung Cancer: Systematic Review

“…This complexity is further compounded by the need to normalize their levels against stable reference miRNAs and the significant discrepancies caused by the different detection platforms, such as qRT-PCR and microarrays. Standardizing the protocols for the selection of reference miRNAs based on the tissue origin and pathological conditions is crucial to increase the reliability of these analyses [21,111,113,114].…”