Tianscript first strand cdna synthesis kit

Total RNA for amplification of P450 cDNA fragments was isolated from the abdomen of adult
D. helophoroidesusing RNAiso Plus (Takara Bio,
www.takarabio.com). Five adult individuals were dissected to remove their abdomens, which then were ground with liquid nitrogen until becoming a powder. The powder was quickly transfered into a 1.5mL centrifuge tube and homogenized with 1mL RNAiso Plus (Takara). The process of total RNA extraction and purification was carried out according to the manufacturer’s instructions. The total RNA (A260/A280 = 1.865) was dissoved in 20 µL DEPC treated H2O and stored at -80°C. The first strand of cDNA was synthesized using 2 µg of the total RNA using a TIANScript First Strand cDNA Synthesis Kit (Tiangen Biotech,
www.tiangen.com) following the manufacturer’s instructions. The reaction mixture was stored at -20°C.

The total RNA was isolated by EASYspin Plus Plant RNA Kit (Aidlab, Beijing, China), and the first-strand cDNA was synthesized by TIANScript First Strand cDNA Synthesis Kit (Tiangen, Beijing, China). The qRT-PCR was performed on the PikoReal real-time PCR system (Thermo Fisher Scientific, CA, USA) with a 10 µL reaction volume, including 5 µL of SYBR Premix ExTaq II (Takara, Dalian, China), 1  µL of cDNA, and 0.2 µL of each primer (Table S3). The reactions were carried out under the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s, and finally end in 20 °C. The protein phosphatase 2A (PP2A) gene of P. mume was selected to be the reference gene for normalization (Wang et al., 2014 (link)). The 2^−ΔΔCt method was used to calculate the relative expression level, and each experiment was repeated in triplicate.

Experimental method for quantitative reverse transcription-PCR was applied as formerly described (Zhou et al., 2016 (link)). The primers for qRT-PCR were designed with Oligo8 software (Supplementary Table 1). Total RNA was extracted from samples using Trizol reagent (Invitrogen, USA) following the manufacturer's instructions. RNA was treated with RNase-free DNase (Promega, USA) to remove residual genomic DNA. Firststrand cDNA was synthesized from 2 μg total RNA using a TIANScript First Strand cDNA Synthesis Kit (Tiangen, China) according to manufacturer's instructions. Realtime RT-PCR was conducted using the PikoReal real-time PCR system (Thermo Fisher Scientific, Germany). Reactions were carried out in a 20 μl volume containing 0.5 μl cDNA, 400 nM each primer, and 10 μl SYBR Premix Ex Taq II (Takara, China) with the following conditions: 30 s at 95°C, 40 cycles of 5 s at 95°C, and 30 s at 60°C. Melting curve analysis was conducted to test the specificity of each primer pair. All qRT-PCR experiments were implemented with three biological replicates, and each replicate was analyzed in triplicate. The relative expression levels were calculated using the 2^−ΔΔCt method, with the protein phosphatase 2A (PP2A) gene of P. mume as the reference gene (Wang et al., 2014 ).