Effects of Preanalytic Variables on Circulating MicroRNAs in Whole Blood.
Author(s): Zhao H, Shen J, Hu Q, Davis W, Medico L, Wang D, Yan L, Guo Y, Liu B, Qin M, Nesline M, Zhu Q, Yao S, Ambrosone CB, Liu S
Publication: Cancer Epidemiol Biomarkers Prev, 2014, Vol. 23, Page 2643-8
PubMed ID: 25472672 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of storage temperature and duration and freeze/thaw cycling on the levels of microRNA (miR)346 and miR134 in blood collected using the PAXgene Blood RNA system and levels of miR16 in EDTA-plasma.
Conclusion of Paper
miR346 and miR134 levels showed significant decreases when blood specimens from either cancer patients or healthy individuals were subjected to 2 freeze/thaw cycles versus 0 or 1, but levels were unaffected by 24 h of blood storage at room temperature or frozen storage for 1 or 6 months. Plasma levels of miR16 significantly decreased when centrifugation of blood was delayed for 18 h at room temperature or when plasma was subjected to increasing numbers of freeze/thaw cycles but not when plasma was stored in a cryovial at -80°C for 6 months or in a straw in liquid nitrogen for 6 months.
Studies
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Study Purpose
The purpose of this study was to determine the effects of storage temperature and duration and freeze/thaw cycling on the levels of miR346 and miR134 in blood collected using the PAXgene Blood RNA system. miR346 and miR134 were identified and validated as ubiquitously expressed microRNAs that exhibited less than 1.2-fold changes between cancer and control specimens. 7 blood specimens from cancer patients and 7 from age, race, and gender matched healthy individuals were used to evaluate the impact for each of 4 preanalytical variables (24 h room temperature delays prior to RNA extraction, 1 month of frozen storage at -80°C versus -20°C, storage at -20°C for 6 months, and 0-2 freeze/thaw cycles).
Summary of Findings:
A 24 room temperature delay before RNA extraction from whole blood had no effect on the levels of miR346 or miR134 among specimens from cancer patients or healthy individuals. Further, storage of blood for 1 month at -20 or -80°C or for 6 months at -20°C had no effect on miR346 or miR134 levels compared to when RNA was extracted immediately. However, both microRNAs showed significant decreases when specimens from either cancer patients or healthy individuals were subjected to 2 freeze/thaw cycles versus 0 or 1 (each cycle consisted of -80°C for 2 weeks with thawing for 2 h at room temperature).
Biospecimens
Preservative Types
- PAXgene
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Healthy
Cancer
Storage Time at room temperature 0 h
24 h
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
Storage Storage duration 0 h
2 weeks
1 month
6 months
Storage Storage temperature -20°C
-80°C
-
Study Purpose
The purpose of this study was to determine the effects of delayed centrifugation, storage temperature, conditions, and duration, and freeze/thaw cycling on the levels of miR16 in EDTA-plasma. miR16 was chosen as a ubiquitously expressed microRNA in plasma that showed a relatively smaller fold-change between specimens from cancer patients and control individuals than miR134 and miR346 (see previous study). Plasma was obtained from PAXgene blood from 7 cancer patients and 7 age, race, and gender matched healthy individuals.
Summary of Findings:
Plasma levels of miR16 significantly decreased when blood was subjected to delayed centrifugation for 18 h at room temperature or when plasma was subjected to increasing numbers of freeze/thaw cycles. However, no significant differences in miR16 levels were measured between fresh plasma, plasma stored in a cryovial at -80°C for 6 months, and plasma stored in a straw in liquid nitrogen for 6 months.
Biospecimens
Preservative Types
- PAXgene
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Healthy
Cancer
Storage Time at room temperature 0 h
18 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
4 cycles
Storage Type of storage container Cryovial
Straw
Storage Storage duration 0 h
6 months
Storage Storage temperature Liquid nitrogen
-80°C