Effects of sample processing and storage on the integrity of cell-free miRNAs in maternal plasma.
Author(s): Olaya LF, Hyett JA, McLennan SV
Publication: Prenat Diagn, 2017, Vol. 37, Page 744-749
PubMed ID: 28556966 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of tube type, delayed centrifugation, and pregnancy on levels of microRNA (miRNA, miR) in plasma.
Conclusion of Paper
Levels of miRNA determined by Qubit were unaffected by tube type and storage duration. Although levels of miR-525 were stable in blood stored in EDTA tubes for 72 h before centrifugation and miR-526a levels were stable for 24 h, levels of both declined significantly when blood was stored in BCT tubes for 24 h or more. In specimens processed 4 h after collection, levels of all seven miRNAs were comparable between the two tube types and were found at higher levels in pregnant than non-pregnant women. Despite being classified as placental; miR-516-5p, miR-520a, miR-526b, and miR-517 were amplified in the plasma of healthy non-pregnant women, but levels of miR-516-5p and miR-520a were significantly lower in plasma from non-pregnant women than pregnant women.
Studies
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Study Purpose
This study investigated the effects of tube type, delayed centrifugation, and pregnancy on levels of miRNA in plasma. Blood from 12 pregnant women and 9 healthy, non-pregnant women was collected into multiple 5 mL K3EDTA and 10 mL cell-free RNA BCT tubes. Blood was mixed well and then the K3EDTA tubes were stored at 4˚C and BCT tubes at room temperature for 4, 24, 48, or 72 h. At each time-point, plasma was obtained from one tube of each type from each patient by centrifugation at 1600 x g for 10 min. To remove debris, plasma was recentrifuged at 16000 x 10 min and then aliquoted and frozen at -80˚C. RNA was isolated using the mirVana Paris Kit and stored at -30˚. RNA was quantified using the MyQubit miRNA Assay. Levels of miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-525, miR-526a*, and miR-526b and spiked-in cel-miR-139 were quantified using TaqMan miRNA assays.
Summary of Findings:
Levels of miRNA determined by Qubit were unaffected by tube type and storage duration. Six of the seven miRNA amplified in all specimens from pregnant women processed within 4 h of collection but miR-517 wasn’t amplifiable in all specimens from pregnant women. Importantly, levels of all seven miRNAs in specimens processed 4 h after collection were comparable between the two tube types. Despite being classified as placental, miR-516-5p, miR-520a, miR-526b, and miR-517 were amplified in the plasma of healthy non-pregnant women in addition to the plasma of pregnant women, but levels of miR-516-5p and miR-520a were significantly lower in plasma from non-pregnant women (P<0.05). Relative levels of miR-525 did not change when blood was stored in EDTA tubes for up to 72 h but variability increased when stored in EDTA tubes for 48 h and levels declined significantly when blood was stored in BCT tubes for 24 h or more (P<0.5 at 24 h, P<0.005 at 48 and 72 h). Levels of miR-526a were comparable when blood was stored in EDTA tubes for 4, 24, or 72 h before centrifugation but was slightly higher when blood was stored in EDTA tubes for 48 h before centrifugation. In contrast, levels of miR-526a decreased significantly (P<0.05) and variability increased when blood was stored in BCT tubes for 24 h or more before centrifugation.
Biospecimens
Preservative Types
- None (Fresh)
- Streck/BCT
Diagnoses:
- Normal
- Pregnant
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Pregnant
Not pregnant
Real-time qRT-PCR Specific Targeted nucleic acid miR-516-5p
miR-517*
miR-518b
miR-520a*
miR-525
miR-526a*
miR-526b
Storage Storage duration 0 h
24 h
48 h
72 h
Biospecimen Acquisition Type of collection container/solution EDTA tube
BCT tube