*4.6. miRNA Quantification*

Total RNA isolation was performed using 200 μL of serum and a miRNeasy serum/plasma advanced kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Elution was performed using 20 μL of RNAse-free water. The successful isolation procedure was confirmed by adding spike-ins and subsequent quantification of these spike-ins.

miRNAs *miR-29c*, *miR-126*, *miR-146a*, *miR-150*, *miR-155*, and *miR-223* were analyzed using the miRCURY LNA miRNA PCR system (Qiagen, Hilden, Germany). As the reference genes, *miR-103a-3p*, *miR-191*, and *miR-423* were used according to the manufacturer's instruction. The possibility of hemolysis was excluded by quantifying *miR-23a* and *miR-451a*. All the reagents were from Qiagen, except where otherwise indicated. qPCR was carried out using Rotor Gene Q.

For reverse transcription, a miRCURY LNA RT Kit was used in a 10 μL reaction master mix containing 2 μL of total RNA, according to the manufacturer's instructions. The resulting reverse transcription was diluted 20-fold and 3 μL was used in a 10 μL reaction master mix, according to the manufacturer's instructions. All the qPCR reactions were performed in duplicate. Prior to qPCR, RNA samples were pooled, followed by RT and qPCR as described above. Efficiency was tested for each analyzed miRNA using 10-fold dilutions and qPCR was performed in triplicate. The signal was collected at the endpoint of every cycle. Following amplification, melting curve analysis of PCR products was performed to verify the specificity and identity. Melting curves were acquired on the SYBR channel using a ramping rate of 0.7 ◦C/60 s for 60–95 ◦C.
