Sybr green 2 master mix reagent kit

Total RNA was extracted from HEK293T and C2C12 myoblasts using TRIzol reagent (Takara; Japan) according to the manufacturer's instructions. For mRNA quantification, 1 μL of total RNA (1000 ng/μL) was reverse transcribed into cDNA using the PrimeScript RT kit (Takara; Japan) and then quantified on an ABI Step One Plus Real-Time PCR system (Applied Biosystems; USA) with SYBR Green II Master Mix Reagent Kit (Takara; Japan) according to the manufacturer's protocols. For miR-487b-3p quantification, a miRNA stem-loop primer and a pair of primers (Table 1) were designed for miRNA reverse transcription and RT-qPCR, respectively. The miR-487b-3p stem-loop was reverse transcribed using the PrimeScript RT reagent kit and quantified by RT-qPCR using SYBR Green II Master Mix Reagent Kit. β-actin and 18S-rRNA were used as internal normalization controls. The cycling conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, and then 60°C for 1 min. Relative gene expression was determined using the 2^-ΔΔCt method.