Total RNA was isolated from the serum using the total RNA purification kit. Briefly, RNA was isolated from 150 μl of serum using the RL buffer washed and eluted in RNAse free water. RNA concentration and purity were quantified using NanoDrop ND-2100 (Thermo Fisher Scientific, USA). To normalize between samples, 3.5 μl Caenorhabditis elegans miR-39 (cel-miR-39) was added to each sample. Immediately after RNA isolation, 5 μl of RNA was reverse transcribed using the microScript microRNA cDNA synthesis kit. cDNA was PCR-amplified (StepOnePlus instrument, Applied Biosystems, USA) using RealQ Plus Master Mix Green, high ROX and miRNA-specific primers for miR-126 and miR-27a. All samples were assayed in duplicate. Relative expression level for a given miRNA was normalized to cel-miR-39 as external control. The expression was calculated as fold change according to the formula: fold change = 2–ΔΔCT in which ΔΔCT = [(CT gene − CT cel − miR − 39)treatment–[CT gene − CT cel − miR − 39)]CTL [33 (link)]. The forward primer sequences of miRs were as follows: miR-126: 5′-TCGTACCGTGAGUTATAATGCG-3′, miR-27a: 5′-TTCACAGTGGCTAAGTTCCGC-3′, and cel-miR-39: 5′-UCACCGGGUGUAAAUCAGCUUG-3′.
We used a universal primer that was supplied by the company as reverse in the reactions.
We used a universal primer that was supplied by the company as reverse in the reactions.
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