Quantitative Real-Time PCR Protocol

RNA was extracted from ten worms for each RNAi population using TRIzol (Invitrogen, Carlsbad, CA, USA) then purified using an RNeasy Mini Kit (QIAGEN, Germantown, MD, USA) including a DNase treatment. cDNA was synthesized from each RNA pool using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol and priming with random hexamers. Primers for qPCR were designed using Primer3 [46 (link)] and are listed in Table 2.
DjGAPDH and SmedGAPDH were used as housekeeping genes for their respective species. qPCR was performed on an MJ Research PTC-200 thermocycler equipped with a Chromo4 Real-Time PCR Detector (Bio-Rad Laboratories, Hercules, CA, USA), using PerfeCTa® SYBR® Green FastMix® (Quantabio, Beverly, MA, USA). Technical triplicates were run for all reactions within an experiment, and two biological replicates were performed. To analyze primer efficiency, standard curves were obtained using a 1:1:1:1 mix of all cDNA pools for each species, serially diluted. The efficiency for each primer pair was found to be between 87–116%. Analysis of relative expression for the genes targeted by RNAi was performed using the ΔΔCt method, where reported values are the mean of all replicates.