*4.3. RNA Extraction, RT-PCR, and qRT-PCR*

Total RNA was extracted from the plant samples using an RNeasy Mini kit (Qiagen). The RNA was treated with RNase-free DNase (Promega) to remove genomic DNA contamination. RT-PCR was performed using an Avian Myeloblastosis Virus (AMV) One-step RT-PCR kit (Takara, Kusatsu, Shiga, Japan). The gene-specific primers used to analyze the selected genes are listed in Table S1. For qRT-PCR, the RNA was subjected to first-strand cDNA synthesis using an Ace-α kit with Oligo-dT primers (Toyobo, Osaka, Japan). The primer sequences were designed according to sequences from the *Brassica* database (BRAD, http://brassicadb.org/brad/). PCR was performed using SYBR® Green Realtime PCR Master Mix-Plus (Toyobo, Japan) under the following cycling conditions: 30 s at 95 ◦C followed by 30 cycles of 95 ◦C for 5 s, 58 ◦C for 10 s, and 72 ◦C for 15 s.
