Primscript first strand cdna synthesis kit

Total RNAs of plant materials, including apple and Arabidopsis, were isolated using the RNA Plant Plus Reagent Kit (Tiangen Biotech, Beijing, China). Reverse transcription was conducted for single-stranded DNA synthesis using the PrimScript™ First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China), per the manufacturer’s protocol. qRT-PCR was performed on the extracted RNA using an ABI7500, in which 18S (apple) and AtACTIN (Arabidopsis) were used as internal control. Then, relative gene expression analysis was conducted using the cycle threshold (Ct) 2^−ΔΔCT method. Quantitative primers are listed in Supplementary Table S1.

Total RNA was extracted from cells in a total RNA Extractor (SanGon, Shanghai, China) following the supplier’s instructions. The quality and quantity of extracted RNA were evaluated using agarose gel electrophoresis and 2,100 Bioanalyzer (Agilent, Germany). The quality-checked RNA was purified and then used as a template to synthesize cDNA first strand followed by cDNA second strand with Primscript™ First-Strand cDNA Synthesis Kit (Takara Bio, United States). PCR amplification was subsequently conducted to establish cDNA libraries. RNA-sequencing was performed on the IlluminaHiseq™ 2,500 platform (http://www.personalmedicine.cn/TechnicalPlatform) in Shanghai Personalbio Technology Co., Ltd.

The extent and mRNA expression levels of TNF-α, IL-6, IL-10, IL-1β, IFN-γ, and TLR4, and NF-κB in each body part of the jejunum, ileum, colon, and spleen and in RAW264.7 cells were clarified by RT–PCR as previously described (31 (link)). Primers for TNF-α, IL-6, IL-10, IL-1β, IFN-γ, TLR4, and NF-κB are described in Table S1. The specific assay steps were as follows: total RNA was extracted from the jejunum, ileum, spleen, and cells by TRIzol reagent (Invitrogen, Carlsbad, CA). Combined with gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), the quality and quantity of total RNA were determined. First-strand cDNA was synthesized from the collected RNA (1 μg) according to the description of the Prim-Script First-Strand cDNA Synthesis Kit (Takara, Otsu, Japan), and the results indicated that the kit is suitable for real-time PCR normalization of the relative amounts of mRNA to GAPDH processing. Then the 2^-ΔΔCt method was used for the next step of analysis.