Plasma miRNA Extraction and Quantification

Total RNA from plasma containing small RNA was extracted using the miRNeasy Plasma Kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of the RNA were determined with a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE). After the synthetization of cDNA using miScript II RT Kit with abidance by the manufacturer's protocol, the performance of qRT-PCR was done by miScript SYBR Green PCR Kit (Qiagen) on a Bio-Rad IQ5 Multicolor RT-PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative levels of miR-340 (Forward: 5′-GCGCGTCCGTCTCAGTTACTT-3′; Reverse: 5′-AGTGCAGGGTCCGAGGTATT-3′) and miR-450b-5p (Forward: 5′-CGCGTTTTGCAATATGTTCC-3′; Reverse: 5′- AGTGCAGGGTCCGAGGTATT-3′) were calculated via the 2^-ΔΔCt method [20 (link)] using miR-16-5p (Forward: 5′-CGCGTAGCAGCACGTAAATA-3′; Reverse: 5′- AGTGCAGGGTCCGAGGTATT-3′) as reference gene, which has been reported to be a marker of hemolysis for its high and stable in the test environment [11 (link), 21 (link)].