Sample Processing and Handling
Pre-analytical variations in sample preparation can compromise the integrity of the circulating miRNAs and the accuracy of the quantification (Kroh et al. 2010; Mcdonald et al. 2011). Removal of all cellular components is an essential and crucial step, as measurements of circulating miRNAs can be confounded by the miR- NAs of haematopoietic cells (Duttagupta et al. 2011; Pritchard et al. 2012). The presence of residual platelets in these preparations is a concern. Platelet contamination may persist in using the standard laboratory protocol for plasma preparation, and hence additional steps of centrifugation are required to eliminate this cellular fraction. This modified protocol was shown to be effective even in samples stored for several years (Cheng et al. 2013).
The choice of anticoagulant can also interfere with miRNA detection. Besides antiplatelet therapy (Cavarretta et al. 2013; De Boer et al. 2013; Kaudewitz et al. 2013; Willeit et al. 2013), intravenous administration of heparin is a confounding factor for miRNA measurements, in particular in patients with cardiovascular disease undergoing percutaneous interventions. Heparin is highly negatively charged and not removed during conventional RNA extraction. It has a dose-dependent inhibitory effect on polymerase chain reaction (PCR) in part due to its binding to magnesium ions (Garcia et al. 2002; Willems et al. 1993; Yokota et al. 1999). Detailed studies demonstrated that heparin has a more pronounced effect on the exogenous Caenorhabditis elegans (C. elegans) spike-in control compared to the endogenous miRNAs, suggesting that alternative ways of normalisation should be applied (Kaudewitz et al. 2013). Citrated or EDTA plasma preparations seem to be a better option, although the induction of haemolysis maybe a concern for citrated plasma (Cui et al. 2011). Apart from platelet activation, erythrocyte haemolysis is an issue, in particular for miRNAs like miR-16 and miR-451 that are highly expressed in red blood cells. Several studies have highlighted the good correlation between the degree of haemolysis and the abundance of these miRNAs in the circulation (Kirschner et al. 2011; Mcdonald et al. 2011).