RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and RT (37°C for 15 min and 85°C for 5 sec, and cooled to 4°C) was performed with a PrimeScript RT Reagent kit (Takara Bio, Inc.) according to the manufacturer's protocols. RT-qPCR was performed (95°C pre-incubation for 5 min, followed by 40 cycles at 95°C for 20 sec, 60°C for 15 sec and 72°C for 20 sec, and cooled to 4°C) on an ABI Prism 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Premix Ex Taq™ (Takara Bio, Inc.). The relative expression of each target gene was quantified by calculating the quantification cycle value, and was normalized to the levels of β-actin. The relative expression levels were calculated based on the 2−ΔΔCq method (19). Each sample was analyzed in triplicate. The primer sequences are listed in Table SII.

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