Total rna extraction reagent kit

For gene expression analysis in SA treatment, tobacco leaves infiltrated with 35S:ECT9-mYFP agrobacterium was sampled for total RNA extraction with and without 2 mM SA treatment. The mYFP expression level is equally regarded as ECT9, and the Basta resistance gene bar was used as an internal control. The A. thaliana mutants were sampled for knock-down genes analysis. All tissues were immediately placed in liquid nitrogen and stored at -80 °C. Total RNA was extracted using the RNA isolation Total RNA Extraction Reagent Kit (Vazyme, R401-01), and 1 µg of total RNA was subjected to reverse transcription using the HiScript^® III 1st Strand cDNA Synthesis Kit (Vazyme, R312-01). Quantitative RT-PCR was performed using a ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) on a CFX Connect Real-Time PCR Detection System (BIO-RAD). UBIQUITIN5 (UBQ5) (AT3G62250) was used as an internal control for sample normalization in the gene expression analysis (2^-ΔΔCt method) (Schmittgen and Livak, 2008 (link)). The primers used are listed in Supplemental Table 1.

Nonparasitized or parasitized P. xylostella larvae were homogenized, and total RNA was extracted with the Total RNA Extraction Reagent Kit (Vazyme) according to the manufacturer’s instructions. The quality and concentration of the isolated total RNA were estimated by electrophoresis and a NanoDrop 2000 (Thermo Fisher Scientific). Then, complementary DNA was synthesized using a PrimeScript 1^st Strand cDNA Synthesis Kit (Takara) according to the manufacturer’s instructions. The entire coding regions of the PxTK, PxTKR and CvBV genes were cloned and inserted into the pGEM-T Easy Vector (Promega). All constructs were sequenced to verify the identity of the sequences. The primers used are listed in S1 Table.

Total RNA was extracted from tissue samples (liver, ovary, and jejunal mucosa) using the RNA isolator Total RNA Extraction Reagent kit (Vazyme). Subsequently, total cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) with 1 μg of total RNA, followed by RT-qPCR amplification using the ChamQ universal SYBR qPCR Master Mix (Vazyme). Gene primers for qRT-PCR were designed by Primer Premier 6.0 software (Premier Biosoft International, United States), synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China), and used in this study (Table 3). The relative expression of the target gene was analyzed using the 2^−ΔΔCt method, normalized against the geometric mean of the expression of β-actin and GAPDH (39 (link)).