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 “tool” 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.
Urinary Exosomal miRNAs: Best Strategies for Isolation and Analysis
Channa Vajjhala, Sarath;Marzia Rossato;
2012-01-01
Abstract
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 “tool” 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.
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