*4.5. Analysis of Expression Pattern of Biosynthetic Related Genes in Seedlings Subjected to LED Light Irradiation*

Approximately 100 mg of powdered leaf samples of five or more seedlings were utilized for total RNA extraction using an RNeasy Plant Mini kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from DNA decontaminated total RNA (5 µg) of each treatment using amfiRivert cDNA Synthesis Platinum Master mix (GenDepot, Katy, TX, USA). The diluted cDNA (1:10) was used as a template, along with gene-specific primers (Table S1) and AccuPower 2× GreenStar Master Mix (Bioneer, Daejeon, Korea), for relative quantification of saponarin, policosanol, and general flavonoid biosynthesis-related genes in quantitative RT-PCR with CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). *HvActin* was used as an internal control. The relative quantification of gene expression was calculated using the 2-∆∆CT method.
