Sybr green real time pcr master mixture

Total RNA was extracted from different developmental stages of F. occidentalis using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. An experimental unit consisted of approximately 100 larvae, pupae, and adults. The extracted RNAs were quantified using a spectrophotometer (NanoDrop, Thermo Fisher Scientific). RNA extract (100 ng per reaction) was used for cDNA synthesis with an RT-premix (Intron Biotechnology, Seoul, Korea). Quantitative PCR (qPCR) was performed using SYBR Green Real-Time PCR master mixture (Toyobo, Osaka, Japan) on a Real-Time PCR System (Step One Plus Real-Time PCR System, Applied Biosystems, Singapore). The reaction mixture (20 μL) contained 10 pmol of gene-specific primers (Table S2) used in RT-PCR and 80 ng of cDNA template. After activating Hotstart Taq DNA polymerase at 94 °C for 5 min, the reaction was amplified with 40 cycles of denaturation at 94 °C for 30 s, annealing at a specific temperature depending on primers (Table S2) for 30 s, and extension at 72 °C for 30 s. The target gene expression levels were normalized to those of EF1, a reference gene. Each treatment was replicated with three independently prepared biological samples. Quantitative analysis was performed using the comparative CT (2^−ΔΔCT) method^{41 (link)}.

Synthesized cDNAs were used for PCR amplification with Cp-TSP13 forward and reverse primers (Cp-TSP13 FP and Cp-TSP RP, Table 2). After an initial denaturation at 94°C for 5 min, RT-PCR was performed with 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min. The PCR reaction was ended with an extension step at 72°C for 10 min. SYBR Green Realtime PCR master mixture (Toyobo, Osaka, Japan) was used for qPCR with a 7500 real time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instruction. The reaction mixture (20 μL) included 5 pmol of forward and reverse primers as described in RT-PCR using 50 ng of template cDNA. After activating Hot-start Taq DNA polymerase at 94°C for 15 min, the reaction was performed with 35 cycles of 30 sec at 94°C, 30 sec at 50°C, and 1 min at 72°C with a final extension for 5 min at 72°C. Fluorescence values were measured and amplification plots were generated in real time with an Exicycler^TM program. In each run, single PCR products were confirmed by their melting curves. Quantitative analysis of amplification was performed using comparative C_T method [73 (link)]. For an endogenous control ribosomal protein (RL32) primers (FP: 5′-ATGCCCAACATTGGTTACGG-3′, RP: 5′-TTCGTTCTCCTGGCTGCGGA-3′) was used.