Pre-Analytical Handling Conditions and Small RNA Recovery from Urine for miRNA Profiling.
Author(s): Armstrong DA, Dessaint JA, Ringelberg CS, Hazlett HF, Howard L, Abdalla MAK, Barnaby RL, Stanton BA, Cervinski MA, Ashare A
Publication: J Mol Diagn, 2018, Vol. 20, Page 565-571
PubMed ID: 29936254 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of precentrifugation storage duration, storage temperature, and freeze-thaw cycling on the yield and count of extracellular vesicles (EVs), small RNA yield, and/or microRNA (miRNA, miR) profile.
Conclusion of Paper
Although EV size and counts were unaffected by storage of urine from a single patient for up to 10 days at 4˚C, small RNA yields decreased more than 55% when urine was stored for 4 h at 2˚C and when stored at 37˚C for 8 h, but storage at 20˚C for up to 24 h resulted in non-significant decreases. Importantly, the authors state a thread-like precipitate occurred when specimens were stored at 2˚C and its clearance by centrifugation may cause a decrease in EVs. The small RNA banding pattern changed when urine was incubated at 37˚C for 4 h or more and Nanostring analysis of a single specimen showed changes in the expression profile of 65 miRNAs with increased time at 37˚C. The miRNA yields were significantly lower from a urine specimen that was frozen at -80˚C and thawed once than from a fresh specimen and the read counts of the measureable miRNAs were very strongly correlated between the freeze-thawed and fresh specimen. Incubation of the freeze-thawed specimen at 37˚C for 5 min allowed some of the precipitate to resuspend and partially attenuated the effects of freezing on miRNA yield. Mean particle size and particle counts were comparable in healthy patients and those with cystic fibrosis.
Studies
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Study Purpose
This study investigated the effects of storing urine from a healthy patient for up to 10 days at 4˚C on the size and count of EVs and also compared results in urine obtained from cystic fibrosis (CF) patients and healthy donors. Urine from a single healthy donor was stored at 4˚C for 1, 2, 3, 6, or 10 days before urine processing and extracellular vesicle isolation. To investigate the effects of CF , EV size and yield were compared in urine collected from nine healthy donors and eight patients with CF. Urine was processed by centrifugation at 200 x g for 10 min and then at 1000 x g for 10 min. Cell-free urine was then stored at -80˚C until exosome isolation using a modified protocol of the Norgen Exosome RNA Isolation Kit. Extracellular vesicles were visualized by TEM and analyzed by nanoparticle tracking analysis.
Summary of Findings:
Extracellular vesicle size and counts were unaffected by storage of urine from a single patient for up to 10 days at 4˚C. Mean particle size and particle counts were comparable in healthy patients and those with cystic fibrosis.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Cystic Fibrosis
- Normal
Platform:
Analyte Technology Platform Cell count/volume Light scattering Cell count/volume Electron microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Cystic fibrosis
Healthy
Storage Storage duration 0 days
1 day
2 days
3 days
6 days
10 days
-
Study Purpose
This study investigated the effects of precentrifugation storage duration, storage temperature, and freeze-thaw cycling on small RNA yield and microRNA (miRNA, miR) profile. To investigate the effects of storage temperature before processing on small RNA yield, urine from a healthy donor was stored for 0, 4, 8, or 24 h at 2˚C and at 20˚C from another healthy donor. To investigate the effects of freeze-thaw on small RNA yield and miRNA profile, a single urine specimen from a patient with CF was stored at -80˚C and then thawed and analyzed immediately or after 5 min at 37˚C. To investigate the effects of diaper collection on small RNA yield and miRNA profile, whole urine from one patient with CF was stored at 37 ˚C for up to 8 h and then at 2-4 ˚C for 6 h before processing. Urine was processed by centrifugation at 200 x g for 10 min and then at 1000 x g for 10 min. Cell-free urine was then stored at -80˚C until exosome isolation using the Norgen Exosome RNA Isolation Kit. Small RNA were quantified and integrity assessed by Bioanalyzer. miRNAs were quantified by real-time PCR assays as well as using Nanostring.
Summary of Findings:
Small RNA and miRNA yields decreased more than 55% when urine was stored for 4 h at 2˚C (P<0.0001) but did not decrease further with additional pre-centrifugation storage. Importantly, the authors state there was a precipitate with thread-like structures that occurred within 2 h when specimens were stored at 2˚C. Centrifugation cleared the precipitate, but, as the precipitate was associated with EVs, the authors state its clearance may cause a decreased in EVs. When urine was stored at 20˚C, a non-significant trend toward decreased small RNA yields was noted but, even after 24 h, the change was limited to 18%. Although the miRNA yields were significantly lower from a urine specimen that was frozen at -80˚C and thawed once than from a fresh specimen (P<0.0001), the read counts of the measureable miRNA were very strongly correlated between the freeze-thawed specimen and the fresh specimen (r=0.99, P<0.0001). Incubation of the freeze-thawed specimen at 37˚C for 5 min allowed some of the precipitate to resuspend and partially attenuated the effects of freezing on miRNA yield. miRNA yields decreased with increased storage duration of urine at 37 ˚C (from 6873-8326 µg/L at 0 h to 2256-2981 µg/L at 8 h). The small RNA banding pattern changed when urine was incubated at 37˚C for 4 h or more and Nanostring analysis of a single specimen showed changes in the expression profile of 65 miRNAs with increased time at 37˚C. Mean particle size and particle counts were comparable in healthy patients and those with cystic fibrosis.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Cystic Fibrosis
- Normal
Platform:
Analyte Technology Platform RNA DNA microarray RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 h
4 h
8 h
24 h
Storage Post-thaw duration 5 min at 37˚C
0 min at 37˚C
Storage Freeze/thaw cycling 0 cycles
1 cycle
Storage Storage temperature 2˚C
20˚C
37˚C