Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations.
OBJECTIVE
The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles.


METHODS
Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times).


RESULTS
Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples.


CONCLUSIONS
This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.
Department of Cardiology, the Heart Centre, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Charlotte
C
Glinge
Department of Medicine I, University Hospital Munich, Campus Grosshadern, Ludwig-Maximilians University Munich (LMU), Munich, Germany.|Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA, United States of America.|DZHK (German Centre for Cardiovascular Research), Partner Site Munich, Munich Heart Alliance (MHA), Munich, Germany.
Sebastian
S
Clauss
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
Kim
K
Boddum
Department of Cardiology, the Heart Centre, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Reza
R
Jabbari
Department of Cardiology, the Heart Centre, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Javad
J
Jabbari
Department of Cardiology, the Heart Centre, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Bjarke
B
Risgaard
Department of Medicine I, University Hospital Munich, Campus Grosshadern, Ludwig-Maximilians University Munich (LMU), Munich, Germany.|DZHK (German Centre for Cardiovascular Research), Partner Site Munich, Munich Heart Alliance (MHA), Munich, Germany.
Philipp
P
Tomsits
Department of Medicine I, University Hospital Munich, Campus Grosshadern, Ludwig-Maximilians University Munich (LMU), Munich, Germany.
Bianca
B
Hildebrand
Department of Medicine I, University Hospital Munich, Campus Grosshadern, Ludwig-Maximilians University Munich (LMU), Munich, Germany.|DZHK (German Centre for Cardiovascular Research), Partner Site Munich, Munich Heart Alliance (MHA), Munich, Germany.
Stefan
S
Kääb
Department of Medicine I, University Hospital Munich, Campus Grosshadern, Ludwig-Maximilians University Munich (LMU), Munich, Germany.|DZHK (German Centre for Cardiovascular Research), Partner Site Munich, Munich Heart Alliance (MHA), Munich, Germany.
Reza
R
Wakili
Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
Thomas
T
Jespersen
Department of Cardiology, the Heart Centre, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Jacob
J
Tfelt-Hansen
D015415:Biomarkers / Q000097:blood; D001800:Blood Specimen Collection / Q000379:methods; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics; D020871:RNA Stability; D013696:Temperature
D015415:Biomarkers; C544547:MIRN1 microRNA, human; C524940:MIRN21 microRNA, human; C532045:MIRN29a microRNA, human; D035683:MicroRNAs