Total RNA was extracted from the buds of C. oleifera using the TRIZOL reagent (Takara, Japan) (Du et al., 2022 (link)). cDNA was synthesized by reverse transcription, and the cDNA samples were mixed with SYBR Green PCR Real Master Mix (Tiangen, China) and 10 μmol/L of each primer. Applied Biosystems 9700 (ABI, USA) was used to conduct PCR. The program settings were as follows: heating for 15 min at 95°C, followed by 42 cycles of 32 s at 95°C, 30 s at 59°C, and 48 s at 67°C. The Primer Quest online software was used to design the qRT-PCR primers (Table S2). The fluorescence signal was collected at the 67°C elongation step of each cycle, and relative quantification was achieved using the 2−ΔΔCt method (Schmittgen and Livak, 2008 (link)). Actin was selected as the internal standard. Three replications were performed on each sample.
Free full text: Click here