Impact of long-term storage and freeze-thawing on eight circulating microRNAs in plasma samples

• Published in: PloS one 2020-01, Vol.15 (1), p.e0227648
• Main Authors: Matias-Garcia, Pamela R, Wilson, Rory, Mussack, Veronika, Reischl, Eva, Waldenberger, Melanie, Gieger, Christian, Anton, Gabriele, Peters, Annette, Kuehn-Steven, Andrea
• Format: Article
• Language: English
• Subjects: Adult; Agent Orange; Biological markers; Biological Specimen Banks - standards; Biology and life sciences; Biomarkers; Biomarkers - blood; Chronic diseases; Chronic illnesses; Circulating MicroRNA - blood; Circulating MicroRNA - genetics; Circulating MicroRNA - isolation & purification; Cohort Studies; Collection; Computer and Information Sciences; Contamination; Criminal investigation; Critical point; EDTA; Environmental impact; Female; Freeze thaw cycles; Freeze-thawing; Freezing - adverse effects; Gene Expression Profiling - methods; Humans; Liquid nitrogen; Low temperature; Medicine and Health Sciences; MicroRNA; MicroRNAs; MicroRNAs - blood; MicroRNAs - genetics; MicroRNAs - isolation & purification; miRNA; Physical Sciences; Polls & surveys; Population studies; Profiling; Research and Analysis Methods; RNA Stability; Room temperature; Specimen Handling - methods; Statistical analysis; Statistical methods; Storage; Studies; Surveys; Thawing; Time; Transcriptome - genetics; Vapor phases; Variance analysis
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0227648

Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample's life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4°C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freeze-thaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon's miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples.