Robust Serum miRNA Isolation Protocol

To ensure sample quality without erythrocyte miRNAs contamination, the level of haemolysis in all serum samples was assessed by spectrophotometry (NanoDrop 1000, USA) [11 (link)]. No serum sample had A414 reading above a value of 0.2 [13 (link)].
Extracellular RNA was isolated from 300 μL of serum by using miRCURY™ RNA isolation kit for biofluids (Exiqon, Denmark). PCR conditions and all reaction mixes were adopted from the user bulletin by Applied Biosystems (Protocol for Creating Custom RT and Preamplification Pools using TaqMan® MicroRNA Assays).
Briefly, multiplex reverse transcription (RT) was performed with TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, CA, USA) and RT primer pool prepared by mixing 5x RT primers provided in TaqMan MicroRNA Assays (Applied Biosystems, CA, USA). To preamplify all tested miRNAs together, TaqMan PreAmp Master Mix was used with PreAmp Primer Pool prepared from 20x TaqMan MicroRNA Assay (Applied Biosystems, CA, USA). qPCRBIO Probe Mix No-ROX (PCR Biosystems, United Kingdom) and 20x TaqMan MicroRNA Assays (Applied Biosystems, CA, USA) were used to perform RT-PCR of individual miRNAs (RotorGene3000 system Corbett Research, Sydney, Australia). All TaqMan MicroRNA Assays are listed in Supplementary material (Online Resource 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/1246129).