Background: Exosomes are cytoplasm containing vesicles released by many cells that can be found in several biological fluids including urine. Urinary exosomes are released from every segment of the nephron, are detectable in urine, constitutively contain RNA (small RNAs and mRNAs) and harbour unique subset of proteins, reflecting their cellular source. Methods: With the aim to establish the optimal protocol for high throughput analysis of exosomal miRNAs, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dyes, analysis of spectrophotometric parameters and capillary electrophoresis. MiRNAs detection and abundance was determined by RT-qPCR. Results: Among the exosomes isolation methods, Ultrafiltration resulted to be the most suited. The highest exosomal RNA yield quantified by RiboGreen® staining was obtained with the combination of TRI ReagentTM with miRNeasy®, followed by TRI ReagentTM, SeraMirTM, miRCURYTM, mirVanaTM and miRNeasy®; but after a multivariate analysis, SeraMirTM scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analysed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. Conclusions: The selection of appropriate urinary exosomal miRNAs isolation method was dependent on various validation results. Ultrafiltration in combination with SeraMir™ exoRNA columns represents the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes, perfectly suited for further applicative research in the field of miRNAs in kidney physiology and pathology.

Optimizing the purification and analysis of miRNAs from urinary exosomes.

Channa Vajjhala, Sarath;ROSSATO, Marzia;MORANDINI, Francesca;CASTAGNA, Annalisa;PIZZOLO, Francesca;BAZZONI, Flavia;OLIVIERI, Oliviero
2014

Abstract

Background: Exosomes are cytoplasm containing vesicles released by many cells that can be found in several biological fluids including urine. Urinary exosomes are released from every segment of the nephron, are detectable in urine, constitutively contain RNA (small RNAs and mRNAs) and harbour unique subset of proteins, reflecting their cellular source. Methods: With the aim to establish the optimal protocol for high throughput analysis of exosomal miRNAs, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dyes, analysis of spectrophotometric parameters and capillary electrophoresis. MiRNAs detection and abundance was determined by RT-qPCR. Results: Among the exosomes isolation methods, Ultrafiltration resulted to be the most suited. The highest exosomal RNA yield quantified by RiboGreen® staining was obtained with the combination of TRI ReagentTM with miRNeasy®, followed by TRI ReagentTM, SeraMirTM, miRCURYTM, mirVanaTM and miRNeasy®; but after a multivariate analysis, SeraMirTM scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analysed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. Conclusions: The selection of appropriate urinary exosomal miRNAs isolation method was dependent on various validation results. Ultrafiltration in combination with SeraMir™ exoRNA columns represents the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes, perfectly suited for further applicative research in the field of miRNAs in kidney physiology and pathology.
Exosomes; micro RNA; urine
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/634753
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