Cucumber Chitinase Expression Analysis

Total RNA was extracted using the Quick RNA plant isolation kit (Beijing Yueyang Biotechnology Ltd., Beijing, China), and cDNA was synthesized using the OneScript^® cDNA Synthesis kit with AccuRT Genomic DNA Removal (Applied Biological Materials Inc, Vancouver, Canada). qRT-PCR was performed in 96-well plates with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster city, CA, USA) using the One-Step BrightGreen qRT-PCR-Low ROX kit (Applied Biological Materials Inc, Vancouver, Canada). Primers were designed using Primer3Plus, and primer specificity was evaluated with NCBI primer-BLAST against the cucumber genome. The specificity of the amplified product was confirmed by a melting curve. Three biological replicates and 3 technical replicates were performed for each combination of cDNA samples and primer pairs. The cucumber β-actin served as the internal control gene [101 (link)]. The relative expression of target genes was analyzed by comparative CT method [102 (link)]. The cucumber chitinase qRT-PCR primers are listed in Table S8.