Pre-Analytical Handling Conditions and Small RNA Recovery from Urine for miRNA Profiling.
J Mol Diagn
; 20(5): 565-571, 2018 09.
Article
em En
| MEDLINE
| ID: mdl-29936254
ABSTRACT
There are currently no standardized protocols for pre-analytical handling of urine to best preserve small RNA for miRNA profiling studies. miRNA is an attractive candidate as a potential biomarker because of the high level of stability in body fluids and its ability to be quantified on multiple high-throughput platforms. We present a comparison of small RNA recovery and stability in urine under alternate pre-analytical handling conditions and extend recommendations on what conditions optimize yield of miRNA from cell-free urine and urine extracellular vesicles (EVs). Using an affinity slurry for isolation of small RNA from urine, we found that urine samples held at room temperature (20°C) for up to 8 hours before processing yield the highest amounts of intact small RNAs from EVs. Some miRNA is lost from urine samples when held 2°C to 4°C and/or frozen before EV isolation, likely because of EV entrapment in uromodulin precipitates. However, we found that a simple 5-minute incubation of urine containing cold-induced precipitate at 37°C resolubilizes much of this precipitate and results in an increased recovery of EVs and miRNAs. Finally, small RNA integrity can be compromised when whole urine is held at 37°C for as little as 4 hours and is not conducive to efficient miRNA profiling.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Perfilação da Expressão Gênica
/
MicroRNAs
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Fase Pré-Analítica
Limite:
Adult
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article