Urinary Exosomal miRNAs: Best Strategies for Isolation and Analysis

Background & Aims: Urinary exosomes are released from every segment of the nephron and harbor unique subset of proteins and RNA. The abundance of small RNAs in exosomes provides a beneficial \u201ctool\u201d to explore specific microRNAs which, in turn could be helpful as noninvasive clinical biomarkers in cardiovascular pathologies [1]. MicroRNAs are endogenous, small (20-22 nucleotides), non-coding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs [2]. The aim of this study was to compare in detail the available strategies for isolation of urinary exosomal microRNAs in combination with different methods for RNA purification. Methods: Urinary Exosomes were isolated using Ultrafiltration (Nanomembrane Concentrators), and Exoquick-TCTM precipitation reagent [3]. Exosomal RNA was isolated using TRI reagent and various commercial kits (Qiagen, Ambion, miRcury and Seramir). Purified total RNA was quantified using Ribogreen, small RNA quantification was obtained using Agilent Bioanalyzer and target miRNAs/mRNAs validation was done by RT-qPCR specific assays. Results: The combination of ultrafiltration for exosomes isolation; and Trizol-miRNeasy for exosomal RNA proved to be efficient compared with all the other methods. Conclusions: The selection of the appropriate urinary exosomal microRNAs isolation method was dependent on the total RNA yield, RNA purity and microRNA abundance. Further analysis in larger groups is required to reconfirm the efficiency of the developed method

Isolation of Urinary Exosomal miRNAs using various methods

Exosomes are involved in a wide spectrum of physiological mechanisms such as immune system modulation, paracrine functions and cell to cell communications. They can be detected in urine being released from every segment of the nephron. Urinary exosomes (UEs) constitutively contain RNA (microRNAs/mRNA/small RNAs) and harbour unique subset of proteins, reflecting their cellular source. With the aim to establish the best method both for urinary exosomes isolation and for the subsequent RNA profiling analysis, we compared three different UEs isolation methods and six RNA extraction techniques. Exosomal RNA yield, quality and size were assessed respectively by specific staining with fluorescent dyes (Ribogreen\uae, RNA specific quantification kit), spectrophotometric quantification (Nanodrop\uae ND \u2013 1000 spectrophotometer) and capillary electrophoresis (Agilent 2100 Bioanalyzer). All the samples were analysed for detection of selected miRNAs and mRNAs by Real Time PCR specific assays. Based on these results the most reliable and convenient method for UEs miRNAs extraction and analysis was selected. Advantages and drawbacks of each methodology were also discussed