Pikoreal 2

For HRM analysis, fluorescent signals were detected at 0.2 °C intervals, with hold-time for 10 s, between 60–95 °C. Data analysis was performed using PikoReal 2.1 Software (Thermo Fisher Scientific, Walthan, MA, USA).

Total RNA was isolated using TRIzol reagent (15596026, Invitrogen, Waltham, MA, USA) and reverse-transcribed into cDNAs with EasyScript^® First-Strand cDNA Synthesis SuperMix (AE301-02, TransGen Biotech, Beijing, China) according to the manufacturer’s protocols.
The cDNA templates were then subjected to RT–qPCR using the SYBR Green PCR Kit (04913914001, Roche, Mannheim, Germany) according to the manufacturer’s instructions. The assay was performed with a PikoReal 96 PCR System (Thermo Fisher Scientific, Waltham, MA, USA). β-Actin was used as an internal control. The relative expression levels were quantified and analyzed using PikoReal 2.1 software (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of the target genes were determined using the 2^−ΔΔCt method. Each sample was analyzed in triplicate. The primers were all synthesized and purchased from GENEWIZ Biotechnology Co., Ltd. (Suzhou, Jiangsu, China). The primer sequences are listed in Supplementary Table S2.