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

Abstract

Salivary microRNAs (miRNAs) have been recently revealed as the next generation of non-invasive biomarkers for the diagnostics of diverse diseases. However, their short and highly homologous sequences make their quantification by RT-qPCR technique highly heterogeneous and study dependent, thus limiting their implementation for clinical applications. In this study, we evaluated the use of a commercial RT-qPCR kit for quantification of salivary miRNAs for clinical diagnostics. MethodsSaliva was sampled from ten healthy volunteers for a time course analysis. A panel of six miRNA targets (with different sequence homologies) were analysed by one of the most commonly used commercially available RT-qPCR kit. Sensitivity and specificity of the tested miRNA assays were corroborated using synthetic miRNAs. The reliability of all tested assays to differentiate miRNA expression profiles were analysed, to statistically discriminate background noise from intrinsic individual signals. ResultsSignificant variabilities in expression profiles of six miRNAs from ten healthy participants were revealed, yet the poor specificity of the assays offered insufficient performance to associate these differences to biological context. Indeed, as the limit of quantification (LOQ) concentrations are from 2-4 logs higher than that of the limit of detection (LOD), the majority of the analysis for salivary miRNAs felt outside the quantification region. Most importantly, a remarkable number of crosstalk reactions exhibiting considerable OFF target signal intensities was detected, indicating their poor specificity and limited reliability. However, the spike-in of synthetic miRNA increased the capacity to discriminate endogenous salivary miRNA at the LOQ concentrations from those that were significantly lower. ConclusionsOur results demonstrate that comparative analyses for salivary miRNA expression profiles by this commercial RT-qPCR kit are most likely associated to technical limitations rather than to biological differences. In particular, assessment of fundamental parameters including LOD, LOQ and crosstalk of each assay is strictly necessary to interpret observed variations. The standardization of rigorous sample handling and experimental design according to technical parameters of each assay plays a crucial role in reducing data inconsistencies across studies. However, further technological breakthroughs are still required to overcome discrepancies in order to accelerate the translation of salivary miRNAs for clinical applications.