RNA Extraction and RT-qPCR

Total RNA was isolated with TRIzol reagent (Takara) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 5 μg of total RNA using oligo(dT)₁₈ as primers. The cDNA was used as a template in a 20-μL PCR amplification. For RT-quantitative (q)PCR, SYBR Green I was added to the reaction and amplified on a real-time PCR detection system (Roche) according to the manufacturer’s instructions. The melting curve was acquired at the end. The transcript data were calculated by Roche’s software and were normalized using 18S ribosomal RNA as an internal control; the relative expression level was calculated by 2^−ΔΔCt. Each experiment was performed with three replicates. The primers for RT-qPCR are listed in Supplemental Table.