Quantitative RT-PCR Analysis of M. graminicola

Total RNA was extracted from M. graminicola nematodes at different life stages as described previously^{37 (link)} using the TRIzol method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA was synthesized using the ReverTra Ace qRT-PCR RT kit (Toyobo, Osaka, Japan). Quantitative RT-PCR was performed with the primer pair MgqRT-PCR-F/MgqRT-PCR-R (Table 1) and actin genes of M. graminicola were amplified as a reference with the primers Mg-ACT-Q-F/Mg-ACT-Q-R^{34 (link)}. The qRT-PCR reactions were performed on a CFX Connect Real-Time System (BIO-RAD, USA) using THUNDERBIRD qRT-PCR Mix (Toyobo, Osaka, Japan). Three technical replicates for each reaction were performed and three independent experiments were performed under the following thermal cycler conditions: 95 °C for 60 sec, 40 cycles at 95 °C for 15 sec, and 60 °C for 30 sec. The relative changes in gene expression were determined using the 2^−ΔΔCT method^{38 (link)}.