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 q
RT-PCR were used to determine RNA interference efficiency. Primers for q
RT-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 q
RT-PCR Sense Primer, 0.6 μL q
RT-PCR Antisense Primer, 1 μL synthesized cDNA, 2 μL ROX and 5.8 μL RNase-free H
2O (Tiangen). The thermal cycling conditions for q
RT-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.
Jiang X., Xu H., Zheng N., Yin X, & Zhang L. (2020). A Chemosensory Protein Detects Antifeedant in Locust (Locusta migratoria). Insects, 12(1), 1.