Cfx96 real time pcr machine

To extract total RNA, the frozen foxtail millet seedling samples were pestled with liquid nitrogen to powder. Total RNA from all simples were extracted with RNaiso Plus reagent (Takara Biotechnology, Beijing, China). To check the quality of the RNA samples, the ultra-low volume spectrometer (BioDrop, Cambridge, UK) was used to measure the concentration of RNA samples and A260/A280. Select RNA samples with A260/280 ratio between 1.8–2.1 for subsequent RNA reverse transcribed into cDNA. Used PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology, Beijing, China) and follow the manufacturer’s instructions to make RNA reverse transcribed into cDNA, and cleaned the gDNA. We diluted cDNA five times for qRT-PCR.
qRT-PCR was performed on a Bio-Rad CFX96 Real Time PCR machine, using TB Green^®Premix Ex Taq™ II (Takara Biotechnology, Dalian, China). qRT-PCR conditions: 95 ℃ for 30 s (initial denaturation), 95 ℃ for 5 s (denaturation),60 ℃ for 30 s (annealing and extending), step denaturation to annealing and extending for 40 cycles, 60 ℃ to 95 ℃ and increment 0.5 ℃ for 5 s (melt curve). Relative gene expression levels were calculated using the ∆∆Cq method [47 (link)] by Bio-Rad Manager 3.1 software, and ACTIN was used as an internal control. Gene-specific primers were designed using Primer Premier 5.0 software (Supplementary Table S1).

Considering that the colon secretes mucus and covers the surface of intestinal epithelial cells to form a mucus barrier, the colonic mucins expression was tested by quantitative real-time PCR to (26 (link)). Total RNA was extracted from the intestinal segments using TRIzol reagent (Tiangen, China) following manufacturer’s instructions. RNA concentration and quality was determined by Nanodrop 2000. Finally, quantitative real-time PCR was performed in a Bio-Rad CFX96 real time PCR machine with the use of SYBR Green Supermix (Takara, Japan) detection protocol. Reaction program was as follows: the mixtures were incubated at 95°C for 5 min, followed by 40 cycles of 5 s at 95°C, 20 s at 60°C, and finally 30 s at 60°C. Melt curves were obtained by increasing the temperature from 65°C to 95°C in 0.5°C increment for 10 s. GAPDH was used as the housekeeping gene. Raw data were analyzed using the 2^-ΔΔCt method. The list of primers was available in Supplementary Table S1.