Prism 7500 sds

RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. The reaction was carried out using the SYBR kit (Shenggong, Shanghai, China), and the reaction ran for 40 cycles. The expression of miR-194-3p was detected by stem-loop primers. The results were analysed with ABI Prism 7500 SDS software. The primers used are shown in Table 1. GAPDH and U6 were used for internal controls.

Total RNA was extracted from approximately 100 mg of frozen jejunal tissues using the reagent box of Total RNA Kit, according to the manufacturer's instructions. The concentration of RNA was measured by using a spectrophotometer, and the purity was ascertained by the A260/A280 ratio. Total RNA from each sample was converted into cDNA according to the manufacturer's instructions and used for RT-PCR.
SYBR Green I RT-PCR Kit was used to measure mRNA expression of antioxidant genes (Nrf2 and gastrointestinal GPx (GPx2)), tight junctions (occludin and claudin-4) and inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8, and TNF-α) expressed relative to the quantity of the β-actin endogenous control. Rat-specific primers were designed from published GenBank sequences and were synthesized by Sangon (Table 1).
For analyses on an ABI PRISM 7500 SDS thermal cycler, PCR reactions were performed with 2.0 µl of first-strand cDNA and 0.4 µl of sense and anti-sense primers in a final volume of 20 µl. Samples were centrifuged briefly and run on the PCR machine using the default fast program (1 cycle at 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 34 s). All of the PCR reactions were performed in triplicate. The relative gene expression levels were determined using the 2^−ΔΔCt method [53] (link).

Total RNA was extracted from cCFU-Fs or isolated cCFU-F exosomes by RNAzol RT (Invitrogen) following the manufacturer’s instructions. The RNA concentrations were determined by using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Isolated RNAs were polyadenylated using an NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen). qRT-PCR was performed with SYBR Green I (DH Biotech, Shanghai) and Prism 7500 SDS (Applied Biosystems; Thermo Fisher Scientific, Inc.). Amplification was performed at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. For mature miRNA expression, the universal primer provided in the NCodeTM miRNA First-Strand cDNA Synthesis Kit was used with one of the following forward primers:
Mmu-miR221: 5′-GUCAACAUCAGUCUGAUAAGCUA-3′and Mmu-miRU6: 5′-ACACGCAAATTCGTGAAGC-3′.
The relative gene expression values were calculated using the ΔΔCt method (ΔΔCt = ΔΔCt treated- ΔΔCt untreated control) with the equation y = 2-ΔΔCt, and U6 was used as a control.