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Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

Background Arterial calcification is associated with cardiovascular mortality in dialysis patients. Active matrix Gla-protein (MGP) is a vitamin K-dependent inhibitor of arterial calcification. Elevated plasma concentrations of inactive MGP, i.e. dephosphorylated-uncarboxylated MGP (dp-ucMGP), is prevalent in dialysis patients. MGP inactivity might contribute to arterial calcification. We investigated if vitamin K supplementation had an effect on arterial calcification in chronic dialysis patients. Methods In a two-year double-blind, placebo-controlled intervention trial, 48 dialysis patients were randomised to vitamin K (menaquinone-7 (MK-7), 360 µg daily), or placebo. MK-7 in serum and dp-ucMGP in plasma were used to assess vitamin K status. Carotid-femoral pulse wave velocity (cfPWV) and scores of coronary arterial calcification (CAC) and abdominal aorta calcification (AAC) were used to assess arterial calcification. Results Thirty-seven participants completed year one, 21 completed year two. At year two, serum MK-7 was 40-fold higher, and plasma dp-ucMGP 40% lower after vitamin K supplementation compared with placebo (mean dp-ucMGP difference: -1380 pmol/L (95% CI: -2029;-730)). There was no significant effect of vitamin K supplementation on cfPWV (mean difference at year two: 1.2 m/s (95% CI: -0.1; 2.4)). CAC Agatston score increased significantly in vitamin K supplemented participants, but not significantly different from placebo (mean difference at year two: 664 (95% CI: -554; 1881)). AAC scores increased in both groups, significantly so within the placebo group at year 1, but with no significant between-group differences. Conclusion Vitamin K supplementation improved vitamin K status, but did not hinder or modify the progression of arterial calcification in dialysis patients.