Rnase free h2o

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.

Genomic DNA was extracted from venous blood using the standard phenol/chloroform method. The concentration and purity of the genomic DNA were measured using Nanodrop (Thermo Scientific, USA). Tris-EDTA buffer was added to the DNA samples to produce a final concentration of 100 ηg/μL. The DNA samples were maintained at −20°C until use.
Total RNA was extracted from the liquid nitrogen-frozen tissues described above, using Trizol reagent (Invitrogen, USA). After being extracted, total RNA was treated with RNase-free H2O (Tiangen, China). The concentration of the extracted RNA was measured using the Nanodrop2000 (Thermo Scientific, USA). First-strand cDNA was synthesized from 1 μg of total RNA using the PrimeScript1 RT Reagent Kit (Perfect Real-Time) (TaKaRa, Biotechnology Co. Ltd., Dalian, China). The reactions were performed under the following conditions: 42°C for 2 min, 37°C for 15 min, and 85°C for 5 sec. The reactions were then stored at 4°C as described in the manufacturer's instructions.

Total RNAs were extracted from cells by a TRIzol reagent (Invitrogen), and the quality and concentration were evaluated for optical density 260/280 ratio using a Nanodrop 2000 spectrophotometer, and reverse transcriptions were performed by the PrimeScript™ RT Reagent Kit with the gDNA Eraser Kit (TaKaRa Biotechnology, Tokyo, Japan), following the manufacturer’s instructions. qPCR primers (Table 2) were designed by Primer Premier 5 software. The reaction system was 10 μL, including 5 μL of SYBR Green Premix Ex Taq (TaKaRa), 3μL of RNasefree H₂O (Tiangen, Beijing, China), 0.5 μL of forward primer, 0.5 μL of reverse primer, and 1 μL of cDNA. The reaction conditions were as follows—95 °C for 3 min, followed by 95 °C for 10 s, the primer-specific annealing temperature for 20 s, and 72 °C for 20 s; these were then repeated 40 times. The β-actin gene was used as an internal control. Each sample was assayed in triplicates.