Plasma Preparation for miRNA Analysis

Blood was collected in 10 mL K2 EDTA Vacutainer tubes (Becton Dickinson, USA) by jugular venepuncture, using 18G needles (Becton Dickinson) and stored at 4°C. Within two hours of collection samples were centrifuged at 1,900 g for 10 min at 4°C to remove blood cells, and then again at 16,000 g for 10 min at 4°C to remove cellular debris and platelets. The second centrifugation step has been shown to significantly reduce platelet numbers in plasma samples, minimising platelet contamination [22 (link)]. In addition, haemolysis was controlled for by using absorbance at 414 nm and the ‘miR ratio’ (ΔCq between miR-451 and miR-23a) as described previously [23 (link), 24 (link)]. All plasma samples were immediately frozen at -80°C.