Optimizing the purification and analysis of miRNAs from urinary exosomes.
Author(s): Channavajjhala SK, Rossato M, Morandini F, Castagna A, Pizzolo F, Bazzoni F, Olivieri O
Publication: Clin Chem Lab Med, 2014, Vol. 52, Page 345-54
PubMed ID: 24101370 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to optimize analysis of microRNA (miRNA, miR) from urinary exosomes by investigating the effects of freezing urine prior to exosome isolation and by comparing three exosome isolation methods and six RNA extraction methods.
Conclusion of Paper
Exosome isolation by ultrafiltration resulted in the lowest poly distribution index and the highest RNA yield and miRNA detection. Although RNA yield was highest when extraction was with a combination of TRI reagent and miRNeasy, this method resulted in contamination that affected the RT-qPCR efficiency. The amplification efficiency of miR-16 as determined by a dilution curve was highest when extraction was with SeraMir. Frozen storage of urine resulted in decreased RNA yield but did not impact the abundance of miR-16 or Let-7i.
Studies
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Study Purpose
This study investigated the effects of exosome isolation method on exosome yield and size and RNA yield and the effects of RNA extraction method on the yield, size, and quality of miRNA from urinary exosomes. Fresh morning urine was collected from an unspecified number of healthy patients and was stabilized by adjusting the pH to 7.0 and adding protease inhibitor cocktail. Cell and debris were removed through centrifugation at 4˚C for 40 min and filtration with a 0.22µm filter. After removal of cells and debris, specimens were stored at -20˚C for 24 h before exosomal extraction by one of three methods: 1) ultrafiltration: centrifugation at 20,000 x g for 20 min at 4˚C, filtration through a Vivaspin concentrator, centrifugation at 3500 x g for 1 h, and treatment with dithiothreitol (DTT) for 10 min at 37˚C; 2) ultracentrifugation: centrifugation at 20,000 x g for 20 min at 4˚C, ultracentrifugation at 200,000 x g for 1 h at 4˚C, pellets washed in PBS with DTT, and then collected by ultracentrifugation at 200,000 x g for 1 h at 4˚C; 3) exosome precipitation: exosomes were precipitated by overnight incubation with Exo-Quick-TC reagent at 4˚C and centrifugation at 1500 x g for 30 min at 4˚C. Purified exosomes were characterized by dynamic light scattering and through Western blotting for aquaporin 2. miRNA was isolated from exosomes using TRI Reagent, a modification of TRI reagent with miRNeasy, miRNeasy, miRVana miRNA isolation kit, miRCURY RNA isolation kit, or the SeraMir exoRNA columns and was stored at -80˚C. Purified RNA was quantified using NanoDrop and RiboGreen and amplification of hsa-miR-16 and hsa-Let-7i was measured by real-time PCR. The RNA size distribution was determined by capillary electrophoresis on a Bioanalyzer and DNA contamination was measured using Quant-iT PicoGreenR dsDNA Kit.
Summary of Findings:
The nanovesicles isolated by each of the three methods contained aquaporin 2, but not the supernatants, confirming that the exosomes were isolated. The purified nanovesicles ranged from 50-90 nm and while all isolation methods resulted in comparable nanovesicle yields, the best distribution was observed when exosomes were isolated by ultrafiltration. Further, the RNA yield and miRNA detection were highest when exosomes were isolated with ultrafiltration. RNA extracted by each of the six methods was free from DNA contamination, enriched for small RNAs (>80% are 26-40 nt), and did not include ribosomal RNA. RNA yield was highest when extraction was with a combination of TRI reagent and miRNeasy, followed by TRI reagent alone, SeraMir, miRCURY, mirVAna, and miRNeasy. The absorbance spectra differed between extraction methods indicating variable levels of contamination with the most contamination present when extraction was with TRI reagent. Further, the ratios of the two urinary miRNA (hsa-miR-16 and hsa-Let-7i) were not comparable among extraction methods, which the authors suggest contaminants present after extraction may decrease the RT-qPCR efficiency. The amplification efficiency of miR-16 as determined by a dilution curve was highest when extraction was with SeraMir (99.8%) or miRCURY (94.3%) but lowest when extracted using TRI reagent with miRNeasy (79.51%, other methods not assessed).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Western blot Cell count/volume Flow cytometry RNA Fluorometry RNA Spectrophotometry RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Filtration Ultracentrifugation
Precipitation
Analyte Extraction and Purification Analyte isolation method TRI reagent
TRI reagent with miRNeasy columns
SeraMir
miRCURY
mirVAna
miRNeasy
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Study Purpose
This study investigated if exosomal RNA yield is impacted by freezing urine. Exosomes were purified from fresh or frozen urine from an unspecified number of healthy individuals by centrifugation at 20,000 x g for 20 min at 4˚C, filtration through a Vivaspin concentrator, centrifugation at 3500 x g for 1 h, and treatment with dithiothreitol (DTT) for 10 min at 37˚C. RNA was extracted using the SeraMir exoRNA columns and quantified by RiboGreen and real-time PCR.
Summary of Findings:
When urine was frozen before exosome isolation, 40% less RNA was obtained then when the exosomes were isolated from fresh urine. However, the abundance of miR-16 and Let-7i was unaffected by frozen storage of the urine prior to exosome isolation.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 0 cycles
1 cycle
Real-time qRT-PCR Specific Targeted nucleic acid miR-16
let-7i
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)