Mj opticon 2

RNA was isolated from 2,285,000 endothelial cells for each condition, including sprouting cells and regular HMVEC-d at the same passage number. Cell cultures were detached using trypsin and washed using PBS. RNeasy (Qiagen, Valencia, CA), RNAse-Free DNase (Qiagen) and QIAshredder homogenizer (Qiagen) kits were used to isolate RNA according to the manufacturer’s protocol.
Reverse transcription reactions were carried out using 3 μg of RNA from endothelial cells (HMVEC–d – P5) in 20 μl volumes with the RT² PCR Array First Strand Kit (SuperArray) and the corresponding manufacturer’s protocol.
The cDNA was analyzed using the RT² PCR Array (SuperArray), which examines 96 genes related to human endothelial cell biology. Preparation for Real-Time PCR was conducted according to the manufacturer’s protocol and readings were taken on the MJ Opticon 2 (Bio-Rad). Data analysis was conducted using SuperArray’s Excel-based PCR Array template to compare gene expression levels amongst genes.

Total RNA was isolated from leaves using Trizol reagent (Invitrogen) according to manufacturer’s instructions and 5 μg RNA was used for cDNA synthesis usiing RevertAid H Minus Reverse Transcriptase (Thermo). Quantitative PCR (qPCR) was performed using the cDNA and gene-specific primers (see Table S4). Each cDNA was amplified using Green Taq DNA polymerase (GenScript) with EvaGreen Dye (Biotium) and the MJ Opticon 2 (Bio-Rad). Tomato Actin expression was used to normalize expression value in each sample, and relative expression values were determined against 10 mM MgCl₂ using the comparative Ct method (2-ΔΔCt) (Pfaffl, 2001 (link)).

Sample preparation prior to transcript quantification in N. benthamiana and tomato experiments differed slightly. For N. benthamiana, transient expression of GFP or GFP:XopX was performed on different regions of the same leaf using 4 × 10⁸ CFU mL⁻¹ of A. tumefaciens. 24 hpi, GFP and GFP:XopX expressing regions were inoculated with 1 mM MgCl₂ (mock) or 2 × 10⁸ CFU mL⁻¹Pst ΔhrcU. For tomato, Xe strains were inoculated into different leaflets of the same leaf at 2 × 10⁸ CFU mL⁻¹. Leaf tissue was excised and total RNA was extracted using TRI Reagent (Life Technologies, Carlsbad, CA). 5 Pg of RNA was used for cDNA synthesis with poly-T primers and Maxima Reverse Transcriptase (Thermo Scientific, Waltham, MA).
Quantitative PCR of cDNA was performed on MJ Opticon 2 (Bio-Rad, Hercules, CA) using SYBR Green reporter (Thermo Scientific, Waltham, MA) with gene-specific primers (Table 2). Relative fold changes in gene transcript abundance were determined by the comparative C_T method (Livak and Schmittgen, 2001 (link)). C_T values were determined by the average of 2 technical replicates per gene per sample. Relative fold change for each gene was calibrated to the average ΔC_T values from mock (1 mM MgCl₂) treatments with NbPP2A or SlACTIN used as the internal control. Mean 2^−ΔΔCT values ± SD for 4 plants are reported.