Optimizing the purification and analysis of miRNAs from urinary exosomes.

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 harbor unique subset of proteins, reflecting their cellular source. METHODS:With the aim of establishing 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 dye, capillary electrophoresis and analysis of spectrophotometric parameters. 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 Reagent™ with miRNeasy®, followed by TRI Reagent™, SeraMir™, miRCURY™, mirVana™ and miRNeasy®; but after a multivariate analysis, SeraMir™ scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analyzed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. CONCLUSIONS:The selection of appropriate urinary exosomal miRNA 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.

journal_name

Clin Chem Lab Med

authors

Channavajjhala SK,Rossato M,Morandini F,Castagna A,Pizzolo F,Bazzoni F,Olivieri O

doi

10.1515/cclm-2013-0562

subject

Has Abstract

pub_date

2014-03-01 00:00:00

pages

345-54

issue

3

eissn

1434-6621

issn

1437-4331

pii

/j/cclm.ahead-of-print/cclm-2013-0562/cclm-2013-05

journal_volume

52

pub_type

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