4.4 RNA isolation and RT-qPCR analysis
Total RNA was isolated from seedlings using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions, and the extracted RNA was treated with DNase to remove potential genomic DNA contamination. First-strand cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) with approximately 2 μg RNA. RT-qPCR reactions were conducted on a 20 μl total reaction volume containing 10 μl of SYBR Green Real-Time PCR Master Mix (Transgene, Beijing, China) and 2 μl cDNA. The CFX96 Real-Time PCR Detection system (Bio-Rad, Hercules, CA, USA) was used for qRT-PCR analysis. The relative expression levels of genes were normalized to the reference gene ACTIN2 (AT3G18780). For each sample, three biological replicates were performed and each biological replicate contained three technical replicates.