2.6. The cDNA synthesis and quantitative real-time PCR

A total of 1 µg RNA was placed into a total volume of 20 µL reverse transcribed reaction to generate cDNA by using PrimeScript RT reagent Kit (miRNAs) (Takara Bio Inc., Shiga, Japan). Quantitative real-time PCR was used to measure the expression levels of miR-126, miR-143, and miR-145 previously demonstrated to be associated with CHD, which was performed on the Light Cycler 480 real-time PCR system. The SYBR^® Premix Ex Taq™ II kit (Takara Bio Inc., Shiga, Japan) and specific PCR primers of miRNAs (Sangon Biotech Co., Ltd., Shanghai, China) were used for the quantification of miRNAs at 95 °C for 30 s, 96 °C for 5 s, 60 °C for 34 s, 40 cycles; 95 °C for 15 s and 60 °C for 1 min; dissolution curve, 95 °C for 15 s, one cycle. The primer sequences used are listed in Table 1. Experiments were performed in triplicate samples. In our experiment, the detection limit of the Ct value was defined as 40. All samples were normalized to internal controls (U6), and the relative expression level was calculated through 2^−ΔΔCt analysis method.^[23],[24]

MiR: microRNA; RT: reverse transcription.