Exosomes can be detected in urine and are released from every segment of the nephron. Urinary exosomes harbor unique subset of proteins, reflecting their cellular source, and constitutively contain RNA. The presence of large amounts of small RNAs in exosomes suggests that exosomes may contain specific microRNAs that provide valuable information as noninvasive clinical biomarkers in both diagnostic and prognostic areas for patients with renal pathologies [1]. We report a comparative analysis of different methods for the isolation of urinary exosomes and the analysis of their microRNA content. We applied and compared several methodologies, including nanomembrane concentrators and Exoquick-TCTM precipitation reagent [2] for exosome purification; TRI reagent and various commercial kits (Qiagen, Ambion, miRcury and Seramir) for exosomal RNAs extraction. Purified RNA was subjected to standard validation such as nanodrop quantification, ribo-pico green measurements and bioanalyser analysis. Moreover all the samples were checked for microRNAs/mRNA/small RNAs content by RT-qPCR specific assays. Based on these results we selected the most reliable and convenient method for microRNAs extraction from urinary exosomes. The selection of the appropriate exosomal miRNA isolation method was dependent on the validation results in terms of RNA yield and the absence of contaminants.
Isolation of Urinary Exosomal miRNAs: Comparative analysis of different methods
Channa Vajjhala, Sarath;Marzia Rossato;BAZZONI, Flavia;OLIVIERI, Oliviero
2012-01-01
Abstract
Exosomes can be detected in urine and are released from every segment of the nephron. Urinary exosomes harbor unique subset of proteins, reflecting their cellular source, and constitutively contain RNA. The presence of large amounts of small RNAs in exosomes suggests that exosomes may contain specific microRNAs that provide valuable information as noninvasive clinical biomarkers in both diagnostic and prognostic areas for patients with renal pathologies [1]. We report a comparative analysis of different methods for the isolation of urinary exosomes and the analysis of their microRNA content. We applied and compared several methodologies, including nanomembrane concentrators and Exoquick-TCTM precipitation reagent [2] for exosome purification; TRI reagent and various commercial kits (Qiagen, Ambion, miRcury and Seramir) for exosomal RNAs extraction. Purified RNA was subjected to standard validation such as nanodrop quantification, ribo-pico green measurements and bioanalyser analysis. Moreover all the samples were checked for microRNAs/mRNA/small RNAs content by RT-qPCR specific assays. Based on these results we selected the most reliable and convenient method for microRNAs extraction from urinary exosomes. The selection of the appropriate exosomal miRNA isolation method was dependent on the validation results in terms of RNA yield and the absence of contaminants.
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