TY - JOUR
T1 - Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations
AU - Glinge, Charlotte
AU - Clauss, Sebastian
AU - Boddum, Kim
AU - Jabbari, Reza
AU - Jabbari, Javad
AU - Risgaard, Bjarke
AU - Tomsits, Philipp
AU - Hildebrand, Bianca
AU - Kääb, Stefan
AU - Wakili, Reza
AU - Jespersen, Thomas
AU - Tfelt-Hansen, Jacob
PY - 2017/2
Y1 - 2017/2
N2 - Background and aim 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 -80C), 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 -80ÊC 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. Conclusion 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.
AB - Background and aim 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 -80C), 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 -80ÊC 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. Conclusion 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.
KW - Biomarkers/blood
KW - Blood Specimen Collection/methods
KW - Female
KW - Healthy Volunteers
KW - Humans
KW - Male
KW - MicroRNAs/blood
KW - RNA Stability
KW - Temperature
U2 - 10.1371/journal.pone.0167969
DO - 10.1371/journal.pone.0167969
M3 - Journal article
C2 - 28151938
SN - 1932-6203
VL - 12
JO - PLOS ONE
JF - PLOS ONE
IS - 2
M1 - e0167969
ER -