Differential Stability of Cell-Free Circulating microRNAs: Implications for Their Utilization as Biomarkers

Abstract

BackgroundMicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study.MethodsThe levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied.ResultsThe levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles.ConclusionsSerum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.

Circulating Serum Exosomal miRNAs As Potential Biomarkers for Esophageal Adenocarcinoma

“…Exosomes are shed by tumor cells, and can enter the circulation. Significantly, circulating miRNAs in exosomes are more stable than other forms, as they are protected from endogenous RNase degradation [10,11], and have significant potential as disease specific biomarkers.…”

Circulating microRNAs: emerging biomarkers for diagnosis and prognosis in patients with gastrointestinal cancers

“…Furthermore, the authors demonstrated miR-1 serum levels to be independently associated with overall survival. Serum miR-122 levels correlated negatively with MELD score, supporting the hypothesis that patients with high miR-122 serum levels do have a better liver function and survive longer [59]. In addition, Luo et al found miRNA-122a expression to be significantly lower in males, and there was a trend towards lower expression in patients who smoked cigarettes, consumed alcohol, had a family history of HCC or were positive for Hepatitis B virus infection.…”

“…Finally, Köberle and co-workers compared the stability of miRNAs packed in microvesicles versus those bound to proteins after incubation with RNase A or RNase inhibitor. They showed that vesicle-associated miRNAs appeared to be more stable and more resistant towards RNase A treatment compared to proteinbound miRNAs [59].…”

“…better protected from RNase treatment) and non-vesicle associated (i.e. less protected from RNAse treatment) miRNAs to be distinguished [59].…”

“…Kalra et al investigated the density gradient technique compared to two other EV isolation techniques (differential centrifugation coupled with ultracentrifugation and epithelial cell adhesion molecule immunoaffinity pulldown), and showed that OptiPrep TM density gradient separation was superior to the two other approaches for isolating pure populations of exosomes [65]. Finally, regarding the additional purification step of using RNAse treatment, this technique is the least expensive approach, easiest to perform, and can be performed using even smaller sample volumes [59]. However, it should be noted that surface proteins on vesicles can protect RNA from degradation, so in order for RNAse treatment to be effective it should be coupled with protease treatment [66].…”