Cfx96 tough real time pcr detection system

Total RNA from roots and leaves was extracted using TRizol reagent (Sigma Aldrich, Belgium) according to the manufacturer’s instructions. RNA concentration was determined using a Quantus fluorometer (Promega, Netherlands). cDNA of each RNA template was synthesized using the iScript^TM kit (Bio-Rad, Belgium) and diluted five times with nuclease-free water. The quantitative reverse transcription PCR (RT-qPCR) assay was performed using a CFX96 Tough Real-time PCR Detection System (Bio-Rad, Belgium). Each PCR reaction contained 6.3 μL Gotagq^®PCR master mix (Promega, Netherlands), 2 μL cDNA, 0.6 μL each primer (5 μM), and 0.2 μL CXR dye (Promega, Netherlands), and 2.3 μL nuclease-free water. The thermal program was set up as follows: 95°C for 3 min; 39 cycles of 95°C for 10 s, and 60°C for 30 s, followed by a melting curve acquisition from 65 to 95°C with the rate of 0.5°C s^–1. The primers used for all target genes are shown in Supplementary Table 1. Elongation factor 1α (EF-1α) and β tubulin (β-TUB) primers were used as housekeeping genes. Gene expression analysis was done using qBase⁺ software (Biogazelle, Zwijnaarde, Belgium). Fold change was computed by diving the CNRQ values (calibrated normalized relative quantities) of the treated samples by values of the control samples. In each gene, four biological replicates and two technical replicates were done.

Roots and leaves were crushed with liquid nitrogen prior to RNA extraction, using TRizol reagent (Sigma Aldrich, Belgium) according to the manufacturer’s instructions. RNA concentration was determined using a Quantus fluorometer (Promega, the Netherlands). cDNA of each RNA template was synthesized using the iScript^TM kit (Bio-rad, Belgium) and diluted 5 times with nuclease-free water. RT-qPCR assay was performed using a CFX96 Tough Real-time PCR Detection System (Bio-rad, Belgium). Each PCR reaction contained 6.25 µL Gotag^®qPCR master mix (Promega, The Netherlands), 2 µL cDNA, 0.625 µL each primer (5µM), and 0.208 µL CXR dye (Promega, The Netherlands), and 2.292 µL nuclease-free water. The thermal programme was set up as follows: 95 °C for 3 min; 39 cycles of 95 °C for 10 s, and 60 °C for 30 s, followed by a melting curve acquisition from 65 to 95 °C with the rate of 0.5 °C/s. The primers used for all genes are shown in Table S3. Elongation factor 1α (EF-1α) and β tubulin (β-TUB) primers were used as reference genes. Gene expression analysis was done using qBase⁺ software (Biogazelle, Belgium). Fold change was calculated by diving the CNRQ values (calibrated normalized relative quantities) of the infected samples by values of the control samples.