Quantifying Plant Immune Responses

Total RNA from leaf tissue was extracted using a GeneJET^™ Plant RNA Purification Kit (Thermo Scientific^™) following the manufacturer’s instructions. DNase treatment of RNA was performed using DNase 1 solution (ThermoFisher^™). The concentration of RNA was measured using a NanoDrop^™ 1000 (Thermo Scientific^™) and the integrity was visualized on a agarose gel after electrophoresis [23 (link)]. Reverse transcription of total RNA was performed as described previously [28 (link)] using a cDNA Reverse Transcription Kit (Applied Biosystems). The reaction mixture (15 μL containing 2 ng of cDNA synthesized with 300 nM of gene specific primers (Invitrogen^™) (Table 2) and 7.5 μL SYBR Green master mix (BioRad) was loaded onto qPCR reaction plates (Applied Biosystems). The reactions were run on a StepOnePlus Real-Time PCR system according to Applied Biosystems protocols. Using the comparative C_T method (ΔΔC_T) method with GAPDH as a control, the relative expression of four genes of interest (TaPR1, TaPR2, TaPR3 (Chitinase) and TaGlu2) were calculated [28 (link),29 (link)].