2.6. Validation of RNA-Seq Data by RT-qPCR

The same RNA samples used in transcriptome sequencing (A. gossypii living in an 8:16 and 12:12 photoperiod for 5 generations) were used to assess the reliability of sequencing and analysis via RT-qPCR. Following that, we validated the expression of candidate genes (in the continuous 8:16 light–dark cycles) and (switching from the fifth generation to a 12:12 light–dark cycle) treatment. A total of 1% agarose gel electrophoresis was used to confirm the integrity of the RNA. In addition, the RNA concentration was assessed using a NanoDrop2000 Ultra-micro nucleic acid protein analyzer (Thermo Scientific, Waltham, MA. USA). Ten DEGs screened according to the transcriptome data were selected for RT-qPCR, and agosDIMT and agosPPI were selected as reference genes [21]. Primers were designed using NCBI Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome accessed on 22 July 2022 (Table S1), before being synthesized by Tsingke Biotechnology Co. (Beijing, China). The sequencing RNA (1 µg) was used to synthesize the first-strand cDNA (template) using a reverse transcription kit (Vazyme Biotech Co., Ltd., Nanjing, China) and then mixed into a 10 µL reaction system by using a ChamQ SYBR fluorescence quantitative kit (Vazyme Biotech Co., Ltd., Nanjing, China). Fluorescence quantitative conditions were 95 °C for 3 min, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. A melting curve analysis was conducted to verify the specificity of amplification. The relative expression levels were calculated using the 2^−ΔΔCT method [22].