2.7. Quantitative Real-Time PCR (qRT-PCR)

S. cerealella proteasome subunit mRNA expression levels were quantified by qRT-PCR on a Bio-Rad iQ2 thermocycler (Bio-Rad, Richmond, CA, USA) to validate the differentially expressed genes (DEG) databases obtained from RNA-sequencing analysis. cDNA was diluted 1:10, and 2 μL was used as input into a 20 μL reaction solution containing 10 μL of SYBR Green Master Mix (Thermo Fisher Scientific, Main St, Miami, USA), 0.8 μL sense and antisense primers, and 6.4 μL of sterilized ultrapure water. Three technical replicates were performed for each biological sample. The reaction conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. A melting curve was obtained at the end of each reaction. Reactions used glyceraldehyde-3-phosphate dehydrogenase as an internal control. Gene-specific primers were designed using Geneious v. 10.1.2. (File S1). A standard curve was produced for primer specificity and efficiency.