*4.4. RNA Isolation*

The RNeasy Micro Kit (Qiagen, Venlo, The Netherlands) was used with an adapted protocol, to isolate total RNA from 200 μL plasma. In summary, using 800 trizol μL reagent (Invitrogen, Breda, The Netherlands), the plasma/Trizol sample was centrifuged for 15 min (15,000× g) after the addition of 160 μL chloroform. Then, 100% ethanol (1.5 volume) was added to the aqueous phase and transferred to a MinElute Spin column (Qiagen) followed by centrifugation for 15 s (18,000× g). Subsequently, 700 μL RWT buffer and twice 500 μL RPE buffer was used to wash the column. The column was centrifuged (18,000× g) for 15 s after the first two washing steps and 2 min (18,000× g) after the third washing step. 15 μL RNase-free water was added for elution of the RNA.
