F301.5 rotor

Lipid extraction of the isolated OMM and IMM membranes was performed according to Bligh and Dyer (Bligh and Dyer, 1959 (link)), where 0.8 volume of membranes at a total protein concentration of 1 mg/ml (OMM or IMM) were mixed with one volume of chloroform and two volumes of methanol [chloroform/methanol/water mixture (1:2:0.8; v/v/v)]. After thoroughly vortexing, one additional volume of chloroform is added to the suspension and again thoroughly vortexed. Next, one volume of distilled water is added [chloroform/methanol/water mixture at (2:2:1.8; v/v/v)] and after mixing the suspension incubated for 30 min at room temperature. To completely phase separate the aqueous from the organic phase, the suspension is centrifuged for 10 min at 12,000 × g (Beckmann F301.5 rotor) at 4°C and recovered the lower organic phase containing the lipids and dried under a stream of nitrogen and stored until further use.

For each subject, 9 mL of peripheral blood was collected in K3 EDTA tubes (Greiner Bio-one, Austria). Plasma was collected from peripheral blood samples using a double-centrifugation protocol described in our previous studies [45 (link),46 (link)], which involves initial centrifugation at 1600× g followed by a second centrifugation at 16,000× g. Microfuge 22R centrifuge and an F301.5 rotor (Beckman Coulter) were used for centrifugation and plasma separation within 4 h after blood taking. After centrifugation, 4 mL of plasma was collected and preserved with 3.2 mL (0.8×) of Trizol (Life Technologies, Carsbad, CA, USA) before storage at −80 °C [45 (link),46 (link)].
Cell-free RNA was extracted from plasma using our established protocol. In brief, 4.5 mL of preserved plasma with Trizol (Life Technologies, Carsbad, CA, USA) was used and the volume was topped up with Trizol to 5.5 mL. The plasma sample was then mixed with 2 mL of chloroform (Sigma–Aldrich, St. Louis, MO, USA), followed by centrifugation of 12,000× g for 15 min at 4 °C. The aqueous layer with RNA was collected and mixed with 1.5 volumes of absolute ethanol (Sigma–Aldrich, St. Louis, MO, USA) to achieve appropriate binding conditions. Total cell-free RNA was then extracted from the mixture using a miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol [47 (link)].

Eighteen HDs were recruited in this study. For each donor, 15 ml peripheral blood was collected in K3 EDTA tubes (Greiner Bio-one, Austria) and divided to two parts evenly. One portion was centrifuged for 3,500 g, 10 min at 4°C, and 3.2 ml plasma was collected and preserved by 9.6 ml Trizol LS Reagent (Thermo Fisher Scientific, USA) before storage at −80°C. Another portion was centrifuged for 1,600 g, 10 min at 4°C followed by 16,000 g for 10 min at 4°C, and 3.2 ml plasma was collected and preserved in the same way. Microfuge 22R Centrifuge and F301.5 rotor (Beckman Coulter) were used for centrifugation in plasma preparation. Blood processing was done within 4 h after blood draw. All donors were recruited with written informed consent. The study was approved by the Joint Chinese University of Hong Kong and New Territories Easter Cluster Clinical Research Ethics Committee (CREC-2014.224).