Quantitative Gene Expression Analysis

Total RNA was extracted from different samples using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using a PrimeScript^TM RT kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Quantitative reverse transcription PCR (RT-qPCR) analysis was performed using the CFX96 real-time system (Bio-Rad, Singapore) with a SYBR Premix ExTaq^TM Kit (TaKaRa), as described previously [29 (link)]. The primers used in the RT-qPCR are shown in Table S2. The 2^−ΔΔCt method was adopted to calculate the relative expression levels. In this study, B. mori glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) was used as the reference gene. The statistical significance between treatments was analyzed using SPSS Statistics software (V 26.0. IBM, NY, USA). Three biological replicates were used.