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®, RNA specific quantification kit), spectrophotometric quantification (Nanodrop® ND – 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.
Isolation of Urinary Exosomal miRNAs using various methods
Channa Vajjhala, Sarath;Marzia Rossato;
2012-01-01
Abstract
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®, RNA specific quantification kit), spectrophotometric quantification (Nanodrop® ND – 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.
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