Selecting the time point for RNA-Seq using Quantitative real-time PCR (qPCR)

In order to select the most informative time point for RNA sequencing to detect significant transcriptomic responses induced by the inoculation treatment, we initially performed a gene expression analysis of selected marker genes using qPCR. We selected inoculated and mock-inoculated clones of two plant genotypes (resistant 193_2.1 and susceptible 1553_5.1), and samples from all three time points (24, 48 and 72 hours post inoculation), resulting in 12 samples. Primers were designed with Primer3 (Rozen & Skaletsky, 1999) based on previously in situsequenced transcriptome of P. lanceolata (unpublished data) and known disease-induced genes in Arabidopsis. We tested seven putative disease-induced genes (Supplementary File 1). Amplification efficiencies (E) of the primer pairs were determined with five dilutions (1 : 1, 1 : 4, 1 : 24, 1 : 124, 1 : 624) of template cDNA, where E = 10-1/slope. The qPCR was performed with three technical replicates, one water control and a plate control sample in a 384-well plate with 10 μL volume, using C1000™ Thermal Cycler (Bio-Rad). All samples were tested for genomic DNA contamination with -RT controls prior to qPCR. Each reaction had 1 μL of the 1:4 diluted cDNA, 5 μL of SYBR® Green containing master mix (iQ™ SYBR® Green Supermix for qPCR; Bio-Rad), 3 μL of nuclease-free water and 0.5 μL (10 µm) of each primer. The cycle conditions were one cycle initiating at 95°C for 3 min, 40 cycles at 95°C for 10 s, 60°C 30 s, and ending with melting curve analysis. To normalize the qPCR data several different reference genes were tested and Elongation factor_CL4, GADPH_28221 and Actin_34737 were validated with geNorm to have stable expression in the samples used in this study (Supplementary File 1). Calculation of relative expression (CNRQ) and normalization was done in qBase+ 3.2.