Hyperscript reverse transcriptase kit

Complementary DNA (cDNA) was obtained from the isolated miRNAs using the HyperScriptTM Reverse Transcriptase kit (GeneAll Biotechnology, Korea). Reverse transcription was performed using SimpliAmp Thermal Cycler (Waltham, MA, USA). Quantitative real-time PCR reactions (qRT-PCR) were performed with the high-capacity StepOnePlus real-time PCR System (Waltham, MA, USA). The thermal cycling conditions were as follows: an initial denaturation step at 95 ℃ for 10 min, 40 cycles of PCR amplification at 95℃ for 15 s, and 60℃ for 1 min, followed by a melting curve analysis program according to the instrument documentation. All real-time PCR reactions were run in triplicate. The sequences of all the primers used are listed in Table 1. The expression levels of miR-29c, miR-31, and miR-18a were examined using the real-time PCR technique and the SYBER Green method, using U6 snRNA as an internal control (housekeeping gene). The cycle threshold (CT) of investigated primers was determined and normalized to the housekeeping gene, RNU6. Fold change of each miRNA expression was calculated using the equation 2^−ΔΔCt.

Peripheral blood samples taken into EDTA tubes were centrifuged for 10 min at 2500 rpm within 2 h. The plasma was separated into Eppendorf tubes and stored at –80 °C until use. miRs from the plasma samples were isolated using a Hybrid-RTM miRNA isolation kit (GeneAll Biotechnology, Korea) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from the isolated miRs using the HyperScriptTM reverse transcriptase kit (GeneAll Biotechnology, Korea). Reverse transcription was carried out using a SimpliAmp thermal cycler (Thermo Fisher Scientific Inc.,Waltham, MA, USA). Quantitative real-time polymerase chain reaction (PCR) reactions (qRT-PCR) were performed using a high-capacity StepOnePlus real-time PCR System (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. All experiments were performed in triplicate. The expression levels of miRs were calculated using the 2-∆∆Ct method.