RT-qPCR for Gene Expression Analysis

Seedlings were grown under specific light photoperiod conditions (12 h light/12 h dark, 16 h light/8 h dark, or 8 h light/16 h dark; light intensity, 200 μmol m⁻² s⁻¹) for 10 d, and samples were harvested at 4-h intervals during a 24-h period. Total RNA was extracted using TRIzol Reagent (Life Technologies) as described by the manual. One microgram of RNA was used for reverse transcription with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). qPCR was performed using SYBR Green Real-Time PCR Master Mix (Toyobo) according to the manufacturer’s instructions on a QuantStudio 3 instrument (Applied Biosystems). The following PCR program was used: 95°C for 2 min, followed by 40 cycles at 95°C for 15 s, 55°C for 15 s, and 72°C for 15 s, followed by a melting-curve analysis. Gene expression was normalized by the geometric mean of ACTIN2 and TUB4 expression as previously described (Li et al., 2019). Experiments were repeated with at least two biological and two technical replicates. Data represent the means ± sd of two technical replicates. Primers used for qPCR are listed in Supplemental Table S1.