Urinary exosomes are 40-100 nm membrane vesicles deriving directly from the kidney and the urogenital tract potentially via each epithelial cell facing the renal tubule lumen. They are released into urine and account for 3% of the total urinary proteome and may also be enriched in specific miRNAs related to renal homeostasis and pathologies. In this context urine can be very helpful as non-invasive source of selected population of exosomal miRNAs. We compared three different urinary exosomes isolation methods and six RNA extraction techniques aiming to establish a reliable method both for urinary exosomes isolation [2] and for the subsequent miRNA profiling [1]. Exosomal RNA yield, size, quality and miRNA detection abundance by Real Time qPCR were assessed. Finally, we could obtain a procedure allowing rapid, accurate and efficient purification and detection of miRNA from urinary exosomes, which is best suited for miRNA rather than large RNA detection. Additionally, the advantages and drawbacks of each different purification strategy were also highlighted. Following our experimental design we ended up with a reliable protocol for miRNAs isolation from urinary exosomes, which should be highly beneficial for further applicative research in the field of kidney biology and diseases.

miRNAs purification and analysis from urinary exosomes.

Channa Vajjhala, Sarath;ROSSATO, Marzia;MORANDINI, Francesca;CASTAGNA, Annalisa;PIZZOLO, Francesca;BAZZONI, Flavia;OLIVIERI, Oliviero
2013

Abstract

Urinary exosomes are 40-100 nm membrane vesicles deriving directly from the kidney and the urogenital tract potentially via each epithelial cell facing the renal tubule lumen. They are released into urine and account for 3% of the total urinary proteome and may also be enriched in specific miRNAs related to renal homeostasis and pathologies. In this context urine can be very helpful as non-invasive source of selected population of exosomal miRNAs. We compared three different urinary exosomes isolation methods and six RNA extraction techniques aiming to establish a reliable method both for urinary exosomes isolation [2] and for the subsequent miRNA profiling [1]. Exosomal RNA yield, size, quality and miRNA detection abundance by Real Time qPCR were assessed. Finally, we could obtain a procedure allowing rapid, accurate and efficient purification and detection of miRNA from urinary exosomes, which is best suited for miRNA rather than large RNA detection. Additionally, the advantages and drawbacks of each different purification strategy were also highlighted. Following our experimental design we ended up with a reliable protocol for miRNAs isolation from urinary exosomes, which should be highly beneficial for further applicative research in the field of kidney biology and diseases.
urine; Exosomes; micro RNA
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/634754
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