RT-qPCR Optimization and Validation

RT-qPCR was performed using the LightCycler® 480 Real-Time PCR System (Roche). Reactions of 25 µl were comprised of 12.5 µl of FastStart Universal SYBR Green PCR Master Mix (Roche, Germany, 2X conc.), 1 µl of each forward and reverse primer (10 µM conc., 0.4  µM final conc.), 5.5 µl of PCR grade water, and 5 µl of cDNA (generally at ~150–170 ng/µl dilutions). The cycling conditions were denaturation at 95 °C for 5 min, followed by 50 cycles of 95 °C for 10 s, 58 °C for 10 s and 72 °C for 10 s. Melting curve analysis was performed after each cycle to ensure primer specificity. All samples were amplified in technical triplicates and a mean value was calculated. Four (M. cerebralis) to five (S. molnari) biological replicates were used for each sample, with the exception of C. shasta (only 3 replicates available). qPCR efficiency was predicted for each gene based on the slope of a linear regression model^{46 (link)} using a series of 5-fold dilutions (1:5, 1:25, 1:125, 1:625). Standard curves were built using Roche Light Cycler 480 Software version 1.5.0 SP4. Generally, for best amplification results efficiency ranges of 90–110% and standard curve slopes of −3.58 to −3.10 were considered optimal^{47 (link)}.