*2.5. RNA Extraction and Quantitative Real-Time PCR Analysis*

Total RNA was extracted using a TransZol Plant kit (TransGen Biotech/TransBionovo, Beijing, China) and the cDNA was synthesized according to the manufacturer's instructions (Takara, Dalian, China). Primers with amplicon lengths of 80–150 bp were designed using Primer5 software. All primer sequences are listed in Table S14. Real-time qRT-PCR was conducted on a Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the SYBR Premix Ex Taq kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. The 10 μL reaction system contained 5 μL of SYBR Green Supermix (2×), 4 μL of cDNA template (30 ng/μL) and 0.5 μL of each primer (10 μM). The qRT-PCR reaction was performed using the following parameters: pre-denaturation at 95 ◦C for 30 s, followed by 39 cycles of denaturation at 95 ◦C for 5 s, annealing at 60 ◦C for 15 s and extension at 72 ◦C for 15 s. The fluorescent signal was measured at the end of each cycle and the melting curve analysis was performed by heating the PCR product from 65 ◦C to 90 ◦C to verify the specificity of the primers. Three independent biological replicates were performed and the qPCR of each replicate was performed in triplicate. The relative expression levels of eggplant *Hsf* and *Hsp* genes were calculated using the 2−Ct method [28]. The *SmEF1a* genes were used as internal controls.
