cDNA was synthesized from 500 ng of total RNA using ReverTra Ace quantitative reverse transcription‑polymerase chain reaction (qPCR) RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) according to the manufacturer's protocol. qRT-PCR was performed in triplicate using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA) and CFX 96 Connect Real-Time System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Thermocycling conditions were 3 min at 95 °C, followed by 40 cycles of 95 °C for 3 s and 60 °C for 20 s. Changes in relative gene expression between cDNA samples were determined using the 2−ΔΔCq method40. The primers used in this study are listed in Supplementary Table S2.
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