Nanodrop nd 2000 uv spectrophotometer

Total RNA was extracted from jejunum and liver tissues using TRIzol reagent and quantified using a NanoDrop® ND-2000 UV spectrophotometer (NanoDrop Technologies, United States). RNA was dissolved in DEPC-treated water and stored at −80°C. Reverse transcription was performed using a PrimeScriptTM RT reagent Kit with gDNA Eraser (TAKARA Bio, Japan), according to the manufacturer’s protocol. Quantitative PCR was performed in triplicate using SYBR Premix Ex Taq (TAKARA Bio, Japan). The target gene expression levels were normalized to that of β-actin. The relative expression level of each target gene was calculated using the 2^−ΔΔCt method, where -ΔΔCT = -(ΔCT_{experimental group} -ΔCT_{control group}) and ΔCT = CT_sample—CT_β-actin. The primers specific for NHE8, NHE3, NHE2, AQP3, AQP4, AGP, CRP, TRF, ALB and β-actin are listed in Table 1.

Total RNA was isolated using Trizol Reagent (Vazyme, Nanjing, China) from snap-frozen jejunal mucosa using the manufacturer’s protocol. The RNA integrity was checked on a 1% ethidium bromide-stained 1.4% agarose formaldehyde gel. Reverse transcription was performed using a commercial kit (PrimeScript RT Reagent Kit; TaKaRa Biotechnology, Dalian, China). The RNA concentration and purity were calculated from the value of OD₂₆₀/OD₂₈₀ (2.1 > ratio > 1.8) using a NanoDrop ND-2000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Total RNA (1 µg) was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s guidelines. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on an ABI StepOnePlus^TM Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA). The cDNA was stored at −20 °C for further determination. The sequence of primers used in this experiment are shown in Table 1. The SYBR Green PCR reaction system was 10 μL in total, which was composed of 5 μL ChamQ SYBR qPCR Master Mix (2×), 0.2 μL forward and reverse primers, 0.2 μL ROX Reference Dye 2 (50×), 1 μL cDNA, and 3.6 μL ddH₂O. The relative mRNA expression of target genes were calculated using the 2^−ΔΔCt method as previously reported [19 (link)]. Primer sequences are shown in Table 1.

Using the EZNA^® Total RNA Isolation Kit (Omega Bio-Tek), RNA was isolate from ileal and liver tissue according to the manufacturer’s protocol. RNA was eluted in DEPC-treated water and stored at -80°C. Total RNA was quantified using a NanoDrop^® ND-2000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States).
Reverse transcription was performed with M-MLV Frist Strand cDNA Synthesis Kit (Omega Bio-Tek), following the manufacturer’s protocol. Quantitative PCR reaction was performed with cDNA temple in triplicate using SYBR Premix Ex Taq (TAKARA Bio, Otsu, Japan). The target genes values were normalize to the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative mRNA level of each target gene were calculated based on the expression of the GAPDH using 2^-ΔCt method. The primers used for qPCR of interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), Toll-like receptor 2 (TLR2), nucleotide-binding oligomerization domain 1 (NOD1), alpha1-acid glycoprotein (AGP), serum amyloid A (SAA), C- reactive protein (CRP), PIT54, ovotransferrin (OVT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are showed in Supplementary Table S2.