Abi 7500 real time quantitative pcr instrument

To validate the RNA-Seq results, several genes with differential expression patterns were confirmed using real-time quantitative PCR. Total RNA was extracted from grains after 14 days of grain filling using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The integrity and concentration of the extracted RNA were detected by agarose gel electrophoresis and scanning with a microspectrophotometer (NanoDrop 2000). Reverse transcription and cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) from 200 ng of total RNA. qRT-PCR was performed following the kits instructions, the first CDNA chain obtained by reverse transcription of 1 μg total RNA was used as the template, and the Fast Start Universal SYBR Green Master Mix with ROX (Roche, Basel, Switzerland) was used in a 20 μL reaction system. Then, the amplification process was completed by an ABI 7500 real-time quantitative PCR instrument (Applied Biosystems, Waltham, MA, USA). The reactions were repeated three times, and the 2^−ΔΔCt method was employed to determine the relative expression levels of the target genes [39 (link)]. The reference gene used was Actin, and some primers were sourced from references [40 (link),41 (link)] and listed in Table S10.

The RNA of ileal tissue was extracted by Trizol according to the instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). Each group had six repetitions. The concentration and quality of RNA were measured by Nano-300 spectrophotometer (Yuanpinghao Biotechnology Co., Ltd., beijing, China). The reverse transcription of RNA was performed by Revert Aid^TM First Strand Gene Synthesis Kit (Takara Biotechnology Co., Ltd., Dalian, China). The real-time quantitative PCR was performed by ABI7500 real-time quantitative PCR instrument (Applied Biosystems Inc, Foster City, CA, USA) and SYBR Premix EX TaqTM II (Takara Biotechnology Co., Ltd., Dalian, China). GAPDH was used as internal reference gene. The relative expression of other genes were calculated according to the former research [19 (link)]. The primer sequences used to determine the expressions of claudin-1, occludin, mucin-2, IFN-γ, TLR2, TLR4, NF-κB, MyD88 and GAPDH were designed by Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast) of NCBI and are listed in the Table 2.