Clin Res Cardiol (2021) DOI DOI https://doi.org/10.1007/s00392-021-01843-w

Impact of repeated freeze-thaw cycles on the stability of miRNAs in human blood samples: implications for biobanking

S. Voss1, C. Troidl2, T. Keller2, C. Lipps2, O. Dörr3, H. Nef3, C. W. Hamm3, C. Liebetrau1

1Abteilung für Kardiologie, Kerckhoff Klinik GmbH, Bad Nauheim; 2Kardiologie und Angiologie, Justus-Liebig-Universität Gießen, Gießen; 3Medizinische Klinik I - Kardiologie und Angiologie, Universitätsklinikum Gießen und Marburg GmbH, Gießen;

Background: MicroRNAs (miRNAs) function as important post-transcriptional regulators of gene expression. In biological fluids (e.g. plasma, serum), miRNAs are emerging as peripheral blood biomarkers in cardiovascular diseases. They are reasonably stable in fluids over time under adequate storage conditions. However, their stability when samples are subjected to repeated freeze-thaw cycles, such as commonly necessary in biobanking, has not yet been determined in detail. Purpose: We aimed to investigate whether repeated freeze-thaw cycles influence the stability of circulating miRNAs in human serum samples. Methods: Peripheral blood samples were collected from n=8 healthy individuals and processed immediately. Serum aliquots were stored at -80°C either under constant storage conditions (control) or they were subjected to repeated freeze-thaw cycles (1 to 5 cycles) within a period of 16 weeks. Serum miRNA expression profiles were determined in controls and after 5 freeze-thaw cycles using miRNA PCR arrays. Quantitative PCR (qPCR) validation of selected, differentially expressed miRNAs was carried out in serum aliquots from all groups (control and 1 to 5 freeze-thaw cycles). Results: PCR array data revealed a high proportion of miRNAs whose amounts were significantly reduced after undergoing 5 freeze-thaw cycles in comparison with controls. Specifically, qPCR analysis confirmed that the levels of 6 miRNAs (let-7b-5p, miR-15b-5p, miR-223-3p, miR-26a-5p, miR-26b-5p, and miR-451a) were reduced after 5 freeze-thaw cycles in comparison with controls (p<0.05 to p<0.001). The level of let-7b-5p was reduced after 2 freeze-thaw cycles (reduction by 34% vs. control, p<0.05), with a subsequent further decrease (reduction by 66% after 5 cycles vs. control, p<0.001). Comparable results were found for miR-15b-5p and miR-26b-5p, showing reduced levels after 3 freeze-thaw cycles and a further decline after 4 and 5 cycles. miR-223-3p and miR-451a were stable up to the 4th freeze-thaw cycle but were reduced after 5 cycles (miR-223-3p: reduction by 58% vs. control, p<0.01, and miR-451a: reduction by 47% vs. control, p<0.05). Conclusion: We demonstrate that repetitive freeze-thaw cycles critically affect miRNA stability in stored serum samples. Since biofluids stored in biobanks are commonly subjected to serial freeze-thaw cycles, standardization of sample handling is important in order to avoid artifacts when measuring circulating miRNAs.

https://dgk.org/kongress_programme/jt2021/aP527.html