Quant cdna synthesis kit

Total RNA was extracted from target tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Reverse transcription was performed using the Quant cDNA Synthesis Kit (Tiangen, Beijing, China) with 1 µg of unpurified total RNA as a template in a 20 μL total volume.
RT-PCR (Promega, Madison, WI, USA) was used to assess the temporal and spatial expression profiles of LmigCSPIII. Primers used are shown in Table 1. The thermal cycling conditions for RT-PCR were as follows: 45 min at 45 °C and 3 min at 95 °C; followed by 30 cycles of 30 s at 95 °C, 30 s at 55 °C and 45 s at 68 °C. The reaction was completed with 10 min at 68 °C.
Both RT-PCR and qRT-PCR were used to determine RNA interference efficiency. Primers for qRT-PCR were specifically designed (Table 1). The actin gene was used as an endogenous control to correct for sample-to-sample variation. The 20 μL reaction system included 10 μL SuperReal PreMix SYBR Green (Tiangen, Beijing, China), 0.6 μL qRT-PCR Sense Primer, 0.6 μL qRT-PCR Antisense Primer, 1 μL synthesized cDNA, 2 μL ROX and 5.8 μL RNase-free H₂O (Tiangen). The thermal cycling conditions for qRT-PCR were 15 min at 95 °C; followed by 40 cycles of 10 s at 95 °C, 20 s at 58 °C and 31 s at 72 °C. Each sample reaction was repeated three times and the results were averaged.

Quant cDNA Synthesis Kit (Tiangen, Beijing, China) was used for reverse transcription of total RNA with 1 μg total RNA as template. Non-quantitative RT-PCRs were performed with gene-specific primers. All the predicted defensins were verified by the conventional RT-PCR with the primers listed in S2 Table (Lmig-ORF 1, 3–5). The integrity of the cDNA preparation was tested with primers for the L. migratoria actin gene [Genebank: AY344445.1]. PCR products were run on 1.2% agarose gels and visualized by ethidium bromide staining. The PCR program began with an initial denaturation at 95°C for 5 min. This was proceeded by 10 cycles of denaturation at 94°C for 30 s, annealing at 68–59°C (-1°C/c) for 30 s, and extension at 72°C for 1 min, which was followed by 20 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min. Then, there was a final extension at 72°C for 5 min.