Potato leaf samples (∼150 mg) were homogenized using a TissueLyzer (Qiagen, Germany) and the total RNA was extracted using RNeasy Plant Mini kits (Qiagen), as described by Baebler et al. (2009) (link). The total RNA from the larval midgut tissue was isolated using TRIzol (Invitrogen, USA), as described by Petek et al. (2012) (link). The RNA concentrations and integrity were validated using a NanoDrop ND-1000 spectrometer (NanoDrop Products, USA) and agarose gel electrophoresis. DNase treatment (DNase I; Invitrogen) and reverse transcription (High Capacity cDNA RT kits; Invitrogen) were performed as described by Baebler et al. (2009) (link). The samples were analysed using a LightCycler 480 real-time PCR system (Roche Applied Science, USA), as described by Petek et al. (2010) (link).
For the potato plants, the analysis included the expression of 26 genes involved in JA biosynthesis and signalling, ET, SA and auxin signalling, phenylpropanoid biosynthesis, gene silencing, photosynthesis, sugar metabolism and genes regulated by phytohormonal pathways. Cytochrome oxidase (COX), elongation factor 1 (EF-1) and 18S rRNA were used as the reference genes for data normalization. In the CPB larvae, 11 midgut-expressed genes involved in larval response to potato defences were assayed, and Ld_smt3 and 18S rRNA were used as the reference genes for data normalization. Detailed descriptions of the assay design and analysis are given in the Supporting Information and in Table S1 (Supporting information). The standard curve approach described by Petek et al. (2012) (link) was used for quantification.
For the potato plants, the analysis included the expression of 26 genes involved in JA biosynthesis and signalling, ET, SA and auxin signalling, phenylpropanoid biosynthesis, gene silencing, photosynthesis, sugar metabolism and genes regulated by phytohormonal pathways. Cytochrome oxidase (COX), elongation factor 1 (EF-1) and 18S rRNA were used as the reference genes for data normalization. In the CPB larvae, 11 midgut-expressed genes involved in larval response to potato defences were assayed, and Ld_smt3 and 18S rRNA were used as the reference genes for data normalization. Detailed descriptions of the assay design and analysis are given in the Supporting Information and in Table S1 (Supporting information). The standard curve approach described by Petek et al. (2012) (link) was used for quantification.
