miRNA Stability in Frozen Plasma Samples.
Author(s): Balzano F, Deiana M, Dei Giudici S, Oggiano A, Baralla A, Pasella S, Mannu A, Pescatori M, Porcu B, Fanciulli G, Zinellu A, Carru C, Deiana L
Publication: Molecules, 2015, Vol. 20, Page 19030-40
PubMed ID: 26492230 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare levels of 8 microRNAs (miRNA, miR) in matched fresh plasma and after storage at -80°C for 6 and 12 months. The authors also compared miRNA levels in fresh plasma with archival frozen plasma.
Conclusion of Paper
Levels of all eight miRNAs investigated were unaffected by ≤ 12 months of storage at -80°C. However, miR-126-3p levels were significantly lower in frozen plasma collected in 1999-2009 than in unmatched fresh plasma (collected in 2013 and promptly analyzed), while miR-126-3p levels were comparable when frozen plasma from 2010 and unmatched fresh plasma were compared. In addition to lower levels of miR-126-3p, plasma collected and frozen in 1999 also had significantly lower levels of miR-125b-5p, miR-425-5p, miR-200b-5p, miR-200c-3p, miR-579-3p and miR-21-5p than unmatched fresh plasma.
Studies
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Study Purpose
The purpose of this study was to compare levels of 8 miRNAs in matched fresh plasma and after storage at -80°C for 6 and 12 months. The authors also compared miRNA levels in fresh plasma with archival frozen plasma. K2EDTA blood was collected in the early morning from five healthy volunteers (30-50 years of age). Plasma was separated by centrifugation at 2500 g for 15 min at 4°C and immediately aliquoted. miRNA was extracted from the fresh aliquot immediately and from the other aliquots after 6 and 12 months of storage at -80°C using the miRNeasy Serum/Plasma Kit. Levels of U6 snRNA, miR-125b-5p, miR-425-5p, miR-200b-5p, miR-200c-3p, miR-579-3p, miR-212-3p, miR-126-3p, and miR-21-5p were quantified by TaqMan real-time RT-PCR assays and levels were normalized to U6 snRNA. Additionally, miRNA was extracted from frozen plasma stored at -80°C since 1999 (5 volunteers), 2002 (4 volunteers), 2003 (5 volunteers), 2009 (5 volunteers), and 2010 (5 volunteers); and miRNA levels from these archival specimens were compared to those in fresh plasma (collected and promptly analyzed in 2013).
Summary of Findings:
Levels of all eight miRNAs investigated were unaffected by ≤ 12 months of storage at -80°C. However, miR-126-3p levels were significantly lower in frozen plasma collected in 1999 (P=0.007), 2002 (P-value not provided), 2003 (P-value not provided), and 2009 (P=0.021), than in unmatched fresh plasma. However, miR-126-3p levels were comparable in frozen plasma collected and frozen in 2010 and unmatched fresh plasma (collected and promptly analyzed in 2013). In addition to the aforementioned reduction in miR-126-3p levels, frozen plasma from 1999 also had lower levels of miR-125b-5p (P=0.007), miR-425-5p (P=0.017), miR-200b-5p (P=0.008), miR-200c-3p (P<0.001), miR-579-3p (P=0.021), and miR-21-5p (P=0.002) in comparison to unmatched fresh plasma.
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 Preservation Type of fixation/preservation Frozen
None (fresh)
Real-time qRT-PCR Specific Targeted nucleic acid U6 snRNA
miR-125b-5p
miR-425-5p
miR-200b-5p
miR-200c-3p
miR-579-3p
miR-212-3p
miR-126-3p
miR-21-5p
Storage Storage duration 0 months
6 months
12 months
2010-2013
2009-2013
2003-2013
2002-2013
1999-2013
Storage Freeze/thaw cycling 0 cycles
1 cycle