Abstract
In the last decade, circulating nucleic acids such as microRNAs (miRNAs) and cell-free DNA (cfDNA) have become increasingly important in serving as potential novel biomarkers for a variety of human diseases. If cell-free nucleic acids are to become routinely used in diagnostics, the difference in plasma miRNA and cfDNA levels between healthy and diseased subjects must exceed pre-analytical and analytical variability. Until now, few studies have addressed the time limitations of pre-processing or explored the potential use of long-term blood storage tubes, which might need to be implemented in real-life diagnostics. In this study, we analyzed the stability of four breast cancer-associated miRNAs and two cancer-associated genes under various storage conditions, to test their limitations for potential application in clinical diagnostics. In two consecutive experiments, we tested the limits of conventional EDTA tubes, as well as long-term storage blood collection tubes (BCTs) from four different manufacturers. We found that circulating miRNAs are relatively stable when stored in EDTA monovettes for up to 12 h before processing. When stored in BCTs, circulating miRNAs and cfDNA are stable for up to 7 days, depending on the manufacturer. Norgen tubes were superior for cfDNA yield, while Streck tubes performed the worst in our study with hemolysis induction. In conclusion, plasma prepared from whole blood is suitable for the quantification of both cf-miRNAs and cfDNA simultaneously.
Highlights
Over the last decade, circulating microRNAs and cell-free DNA have increasingly become of interest as novel biomarkers for a variety of human diseases, including cancer [1,2]
Aside from the cellular form, circulating cell-free miRNAs have been shown to be remarkably stable in body fluids such as plasma, making them a potential non-invasive diagnostic tool for early cancer detection [6,7,8]. cell-free DNA (cfDNA) found circulating in the blood is proposed to stem, in varying ratios, from DNA released from tumor cells together with DNA fragments from normal cells [9]
In order to determine the stability of cf-miRNAs after storage in EDTA monovettes, blood was drawn from six healthy individuals and stored at either room temperature (RT) or 4 ◦C for up to 18 h
Summary
Over the last decade, circulating microRNAs (miRNAs) and cell-free DNA (cfDNA) have increasingly become of interest as novel biomarkers for a variety of human diseases, including cancer [1,2]. Aside from the cellular form, circulating cell-free miRNAs (cf-miRNAs) have been shown to be remarkably stable in body fluids such as plasma, making them a potential non-invasive diagnostic tool for early cancer detection [6,7,8]. A combination of a panel of DNA mutations and proteins detected in plasma was able to detect five different cancer types with a sensitivity of 69–98% and a specificity of >99% [10]. The combinations of cfDNA and cf-miRNA may be powerful as biomarkers in cancer with complementary prognostic values regarding specificity and sensitivity in liquid biopsies
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