Mirneasy serum plasma isolation kit

Venous blood was collected and kept on ice for 45 min. After centrifugation at 2000×g and 4 °C and for 10 min, the supernatant serum was collected and stored in aliquots at − 80 °C until further analysis. Samples with visible haemolysis were excluded. Total small RNAs were extracted from 200 μL serum using the miRNeasy serum plasma isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Synthetic C. elegans (C) miR-39-3p (Qiagen, Hilden, Germany) was spiked-in at a final concentration of 1.6 × 108 copies/μL after the initial denaturation, prior to extraction, to correct the extraction efficiency. The total RNA was eluted in 14 μL of RNase-free water. A fixed volume of 7 μL of eluate was used as input for the cDNA synthesis. RNA was converted to cDNA using the qScript™ microRNA cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD). qPCR was performed with PerfeCta® SYBR® Green Supermix on an Applied Biosystems (Foster City, CA) 7900 Real Time PCR System with the following conditions: 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 15 s and 70 °C for 15 s. MicroRNA expression levels were normalized to the mean of spiked-in miR C-miR-39 and presented as 2^−ΔCt values.

Total RNA from serum/plasma was isolated using miRNeasy serum/plasma isolation kit (Qiagen) according to manufacturer’s protocol. Briefly, 20 µl serum/plasma was added with 180 µl phosphate-buffered saline (PBS). 1 ml QIAzol lysis reagent was spiked with a) 1 µg MS2 carrier RNA (Roche) to reduce loss of RNA during isolation and b) a set of 3 synthetic small RNAs (MiRXES, Singapore) with sequences distinct from any of the 2588 annotated mature human miRNAs (miRBase version 21) to monitor RNA isolation efficiency. This QIAzol mixture was aliquoted to the 200 µl sample for lysis. 200 µl chloroform was then added, thoroughly mixed and centrifuged at 18,000 g for 15 minutes (min). 600 µl aqueous phase was subsequently removed for spin-column based purification and RNA was eluted in 30 µl nuclease-free water.