Stability of Circulating Blood-Based MicroRNAs – Pre-Analytic Methodological Considerations

Abstract

Background and aimThe 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.MethodsBlood 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).ResultsDifferent 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.ConclusionThis 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.

miRNAs in platelet-poor blood plasma and purified RNA are highly stable: a confirmatory study

“…Certain tissue-specific miRNAs in circulation, like miR-1 (muscle) or miR-122 (hepatocytes), have been reported to be more sensitive to freeze/thaw than others (31). Decreased detection of several plasma miRNAs after freeze/thaw cycles (32) or incubation of plasma at room temperature (33) was recently reported. To help resolve these apparent differences, we re-examined stability of several commonly investigated miRNAs in platelet-rich and platelet-poor plasma after incubation of plasma at 22 °C and after various freeze-thaw cycles.…”

MicroRNA Isolation by Trizol-Based Method and Its Stability in Stored Serum and cDNA Derivatives

“…Indeed, there are an increasing amount of studies showing that the stability of miRNAs is varied upon prolong incubation and in different storage conditions, depending on the types of miRNAs. Glinge et al, (2017) found that miR-21 and miR-1 are stable for at least 4 days in EDTA-treated whole blood, whereas only miR-1 significantly degraded within 4 days in serum, separatortreated, whole blood. Some studies have shown that the degradation of miR-1 and miR-122 expeditiously occurred within 3 hrs, whereas other miRNAs declined only slightly within the initial 5 hrs, after serum storage at room temperature (Koberle et al, 2013).…”

“…Chen et al, (2008) has demonstrated that miRNAs in both extracted serum RNAs and serum without RNA extraction are resistant to RNaseA digestion, low/high pH, and 10 freeze-thaw cycles at -80°C (Chen et al, 2008;Mitchell et al, 2008). Likewise, miR-451a and miR-23a in unhemolyzed blood samples, kept at room temperature, and at 4°C are stable for at least 8 hrs (Wu et al, 2016) as well as storage of plasma at -80˚C for 9 months having no significant effect on miR-21 and miR-29b levels (Glinge et al, 2017). Moreover, Grasedieck et al, (2012) have shown that miRNAs, from serum, are stable at -20˚C and -80˚C for 10 days (short-term storage), 20 months (intermediate-term storage), and at least 4 years (long-term storage).…”

“…Some studies have shown that the degradation of miR-1 and miR-122 expeditiously occurred within 3 hrs, whereas other miRNAs declined only slightly within the initial 5 hrs, after serum storage at room temperature (Koberle et al, 2013). In addition, instability of miRNAs is also observed after repeated freeze-thaw cycles for miR-24, miR-93, miR-223, miR-451, and miR-21 when plasma or serum are kept at -20˚C and -80˚C (Grasedieck et al, 2012;Glinge et al, 2017). There are several reasons to explain the inconsistency in these results.…”

“…However, an increasing number of studies have demonstrated that the instability of the miRNAs has been found in various storage conditions. Degradation of miRNAs are obviously seen in stored serum, or plasma at room temperature within 5-24 hrs (Mitchell et al, 2008;Koberle et al, 2013;Wu et al, 2016;Glinge et al, 2017). In addition, the miRNAs are unstable when such samples are stored at 4°C, -20°C, and -80°C within different time periods (Sourvinou et al, 2013;Wu et al, 2016), and the repeated freeze-thaw cycles of these samples also leads to a significant decrease in miRNA levels (Grasedieck et al, 2012;Ge et al, 2014;Zhao et al, 2014;Glinge et al, 2017).…”