Quantitative PCR for Gene Expression

Gene expression differences were determined by real-time quantitative PCR (qPCR) as described before with slight modification [27 (link),28 (link)]. Briefly, Primer Premier 5.0 (PREMIER Biosoft international, Palo Alto, CA, USA) was used for defining specific PxTryp_SPc1 gene primers (Supplementary Materials Table S1). PCR reactions (20 μL) contained 7.4 μL RNase-Free ddH₂O, 10 μL of 2 × FastFire qPCR PreMix Plus (TIANGEN, Beijing, China), 5 μM of each specific primer, 1 μL of first-strand cDNA template, and 0.4 μL 50 × ROX Reference Dye (TIANGEN, Beijing, China). The running program consisted of a denaturation at 95 °C for 10 min followed by 40 denaturalized cycles at 95 °C for 15 s, annealing at 57 °C for 30 s, and extension at 72 °C for 30 s. All reactions were performed in an Applied Biosystems QuantStudio 3 Real-Time PCR System (Applied Biosystems, Forster City, CA, USA). As an internal control for relative quantification, the ribosomal protein L32 (RPL32) gene (GenBank accession no. AB180441) was used in qPCR data analysis. Three biological repetitions and four technical repetitions were conducted for each sample. To define the statistically differences, one-way ANOVAs with Duncan’s test (overall significance level p < 0.05) were used.