Stability of miRNA in human urine supports its biomarker potential.
Author(s): Mall C, Rocke DM, Durbin-Johnson B, Weiss RH
Publication: Biomark Med, 2013, Vol. 7, Page 623-31
PubMed ID: 23905899 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of centrifugation, storage at 4˚C or room temperature, freeze-thaw cycling, and treatment with trypsin on levels of microRNA-16 (miR-16) and microRNA-21 (miR-21) in spot urine from healthy volunteers.
Conclusion of Paper
Levels of miR-16 and miR-21 were much higher in the pellet of centrifuged urine than in the supernatant or in uncentrifuged urine. Levels of miR-16 and miR-21 decreased with each freeze-thaw cycle and during storage at room temperature or 4˚C. However, the ratio of miR-16 to miR-21 was constant over the five days of storage at either temperature. Treatment of urine from five patients with trypsin had no effect on the cycle threshold (CT) of miR-16 or miR-21 in urinary exosomes, indicating their stability is not dependent on association with exosome bound structures
Studies
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Study Purpose
This study investigated the effects of centrifugation, storage for up to five days at 4˚C or room temperature, up to 10 freeze-thaw cycles, and treatment with trypsin on levels of miR-16 and miR-21 in spot urine from eight healthy volunteers. Urine aliquots were stored at 4˚C and room temperature for up to five days or subjected to up to 10 freeze-thaw cycles from -80˚C to room temperature before centrifugation at 25,000 x g at 4˚C and extraction of RNA from the resulting pellet. To investigate the effect of centrifugation, urine from three patients was centrifuged at 25,000 x g at 4˚C for 15 min and RNA was extracted from the pellet and the supernatant and from uncentrifuged aliquots from the same void. RNA was extracted with the miRNeasy Mini Kit and quantified by real-time PCR. Urinary exosomes were isolated from the urine of five healthy individual by a differential centrifugation protocol, but details were not specified.
Summary of Findings:
Levels of miR-16 and miR-21 were much higher (7-9 CT) in the pellet of centrifuged urine than in the supernatant (P<=0.0001 and P=0.0023) or in uncentrifuged urine (P<0.0095 and P=0.0017, respectively). Levels of miR-16 and miR-21 decreased 14% and 10% (respectively), with each freeze-thaw cycle such that only 23% of the miR-16 and 37% of miR-21 remained after 10 freeze-thaw cycles. Similarly, miR16 and miR-21 declined by 19% per day when urine was stored at room temperature and 16% and 11% (respectively) per day when urine was stored at 4˚C. Thus, 35% of miR-16 and miR-21 remained after five days at room temperature, but 42% of the miR-16 and 56% of the miR-21 remained after five days at 4˚C. Importantly, the ratio of miR-16 to miR-21 was constant over the five days of storage at either temperature. Treatment of urine from five patients with trypsin had no effect on the CT of miR-16 or miR-21 in urinary exosomes, indicating their stability is not dependent on association with exosome bound structures.
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Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifuged
Not centrifuged
Biospecimen Preservation Type of fixation/preservation None (fresh)
Frozen
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
5 cycles
6 cycles
7 cycles
8 cycles
9 cycles
10 cycles
Storage Storage temperature Room temperature
4˚C
Storage Storage duration 3 days
4 days
5 days
0 h
1 h
3 h
6 h
12 h
1 day
2 days
Real-time qRT-PCR Specific Targeted nucleic acid miR-16
miR-21