Optimizing miR-29 measurements in biobanked, heparinized samples.
Author(s): Warnement CM, Cismowski MJ, Rogers LK
Publication: Life Sci, 2019, Vol. , Page 116894
PubMed ID: 31626789 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of heparinase treatment, freeze-thaw cycling, and hemoglobin concentration on levels of miR-29 isoforms in plasma.
Conclusion of Paper
miR-29 isoforms were not quantifiable in heparin plasma without heparinase treatment or after treatment with R&D heparinase III for 1 or 2 h, but cycle threshold (CT) values were within 1 cycle of those in EDTA plasma when plasma was treated for 1 or 2 h with R&D heparinase I or II or NEB heparinase I. Levels of miR-29b-3p were non-significantly lower in frozen than fresh specimens but were significantly lower in specimens freeze-thaw cycled 5 or 10 times than in fresh specimens. Levels of miR-29b-3p were not correlated with hemoglobin concentration.
Studies
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Study Purpose
This study investigated the effects of heparinase treatment, freeze-thaw cycling, and hemoglobin levels on levels of miR-29 isoforms in plasma. Plasma was obtained from the blood of three healthy volunteers in EDTA and Heparin tubes (processing details not provided). The effects of heparinase treatment were investigated by incubation of plasma with R&D systems recombinant F. heparinum heparinase I, recombinant P. heparinus heparinase II, and recombinant P. heparinus heparinase III or New England Biolabs (NEB) heparinase I. An aliquot of heparinase-treated plasma was removed after 1 h and another after 2 h. Plasma was analyzed fresh, after one freeze-thaw cycle (at -80°C for 72 h), and again after 5 and 10 freeze-thaw cycles. Hemoglobin levels were determined by ELISA. miR-29a-3p, miR-29a-5p, miR-29b2-5p, miR-29c-3p, and miR-29c-5p were quantified by real-time PCR.
Summary of Findings:
miR-29 isoforms were not quantifiable in heparin plasma without heparinase treatment or after treatment with R&D heparinase III for 1 or 2 h, but CT values were within 1 cycle of those in EDTA plasma when plasma was treated for 1 or 2 h with R&D heparinase I or II, or NEB heparinase. Levels of miR-29b-3p were non-significantly lower in frozen than fresh specimens but were significantly lower in specimens freeze-thaw cycled 5 or 10 times than in fresh specimens (P=0.033 and P=0.020, respectively). Levels of miR-29b-3p were not correlated with hemoglobin concentration.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein ELISA RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant EDTA
Heparin
Biospecimen Aliquots and Components Hemolysis Present
RT-PCR Specific Targeted nucleic acid Target miR-29-3p
Storage Freeze/thaw cycling 0 cycles
1 cycle
5 cycles
10 cycles
RT-PCR Specific Template modification Untreated
Treated with R&D systems recombinant F. heparinum heparinase I (I)
Treated with R&D systems recombinant P. heparinus heparinase II (2)
Treated with R&D systems recombinant P. heparinus heparinase III (3)
Treated with NEB heparinase I
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)