Nanodrop nd 2000 spectrophotometer

The TRIzol reagent (Invitrogen, China) was applied to extracted total RNA. RNA concentration was examined with a NanoDrop ND-2000 spectrophotometer (Life Technologies). Quantitative real-time PCR (qPCR) was carried out by the application of the SYBR Green quantitative PCR kit (Takara, Hangzhou, Zhejiang, China) using the 7500 Real-Time PCR System. The results were normalized to the expression of GAPDH. The primers were as follows: GAPDH sense 5′- ACAACTTTGGTATCGTGGAAGG-3′, reverse 5′- GCCATCACGCCACAGTTTC-3′ and PLXDC2 sense, 5′- CCAGTTTCAGTTCGCCGATG-3′, reverse 5′- TGTCTACCGCCTTGAGAAAGT-3′. The qRT-PCR data were analyzed and calculated using the 2^−ΔΔCt methods.

Total RNA was extracted from various tissues and cells using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instruction. The quality of total RNA was checked by 1% agarose gel electrophoresis. The concentration of total RNA was determined using a Nanodrop ND-2000 spectrophotometer (Thermo Electron Corporation, USA). The first-strand cDNA was generated using the Revert Aid™ M-MLV Reverse Transcriptase Kit (Promega, USA) following the manufacturer’s instructions. The cDNA products were stored at –20°C.

Extraction of the total RNA was done via the RNA easy Extraction Kit (Qiagen) according to the manufacturer’s guidelines. Both yield and purity were assessed at 260 and 280 nm respectively using a Nanodrop ND-2000 spectrophotometer (Thermo Electron). A total amount of 1 μg RNA was used for cDNA synthesis by Viva 2-steps RT-PCR Kit according to the manufacturer’s instructions.
Quantitative PCR using Thermo Scientific Verso 1-step RT-PCR Ready-Mix kit (Applied Biosystems, Foster City, CA, USA) was conducted to analyze the levels of the mRNA of the target genes which quantified relatively in relation to the expression rate of β-actin. The primers of amplification include IL-6 F: 5′-GCCT TCTTGGGACTGATG-3, R: 5′-TGGTCTGTTGTGGGTGGT-3′, TNF-α F: 5′-GCTGAGGTTGGACGGATAAA-3′, R: 5′-AAAATCCTGCCCTGTCACAC -3′, and TGF-β F: 5′-TGGCGTTACC TTGGTAACC- 3′, R: 5′- GGTGTTGAGCCCTTTCCAG- 3′, and B-actin, F: 5-d TCCCTGAAGTACCCCATGGAG-3′, R: 5′-d TTGGCCTTGGGGTTCAGGGGG-3.

Total RNA was extracted using TRIzol Reagent (TRIzol^® Plus RNA Purification Kit; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The integrity and purity of RNA were determined by electrophoresis on a 1% agarose gel and a Nanodrop ND-2000 spectrophotometer (Thermo Electron Corp., Waltham, MA, USA), respectively. Total RNA was reverse-transcribed to cDNA with a PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) and stored at −20 °C before analysis.

Total RNA was extracted from various tissues using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instruction. The quality of total RNA was checked by 1% agarose gel electrophoresis. The concentration of total RNA was determined using a Nanodrop ND-2000 spectrophotometer (Thermo Electron Corporation, USA). The first-strand cDNA was generated using the Revert Aid™ M-MLV Reverse Transcriptase Kit (Promega, USA) following the manufacturer’s instructions. The cDNA products were stored at −20 °C.

To validate the Illumina sequencing data, twenty immune-related DEGs were chosen for quantitative real-time PCR (qPCR) analysis. The integrity and purity of RNA were determined by electrophoresis on a 1% agarose gel and a Nanodrop ND-2000 spectrophotometer (Thermo Electron Corp., Waltham, MA, USA), respectively. Total RNA was reverse-transcribed to cDNA with the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan). The primers were designed with the Primer 5 software (Premier Biosoft International). The β-actin was selected as a reference gene for the qPCR analysis, due to its stably expressed characteristic [25 (link), 26 (link)]. The qPCR was performed with TB Green Premix ExTaqII (TaKaRa, Tokyo, Japan). The reactions were carried out in a total volume of 20 μL containing 2 μL of diluted cDNA (50 μg/μL), 1 μL of each primer, 10 μL of TB Green PCR Master Mix and 6 μL of H₂O, with the following cycling profile: 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Each sample was processed in triplicate in the Roche LightCycler 480 Real-Time PCR System (Roche Diagnostics Burgess Hill, UK). The 2^-ΔΔCT method [81 (link)] was used to analyze the expression level.

Prior to the collection of the fecal samples, none of the subjects had taken antibiotics drugs or probiotic, prebiotic, or symbiotic supplements in the previous 2 months. Obese individuals and those with diabetes, cardiovascular disease, irritable bowel syndrome, inflammatory bowel disease, autoimmune diseases, allergic diseases, or other comorbidities; those who had abused drugs or alcohol in the last year; and those who had known active bacterial, fungal, or viral infections were excluded from the study. Also, patients had a history of gastrointestinal surgery and accepted colonoscopy before 4 weeks ago were not included in our studies.
Fresh fecal samples were collected in a sterile plastic cup at the hospital and refrigerated. The samples for bacterial genomic DNA extraction were delivered to the laboratory within 30 min of collection and stored at −80°C. DNA extractions were performed using a FastDNATM SPIN Kit for Feces (MP Biomedicals) according to the manufacturer's protocol with additional glass‐bead beating steps performed using a Mini‐Beadbeater (Thermo Electron Corp.). DNA was quantified using a NanoDrop ND‐2000 spectrophotometer (Thermo Electron Corp.), and the integrity and size were assessed using 1.0% agarose gel electrophoresis on gels containing 0.5 mg/ml of ethidium bromide. Finally, DNA was stored at −20°C until further use.