Abi stepone rt pcr system

Genes related to phenolic compounds were analyzed by quantitative real-time PCR (qRT-PCR) and the sequences of primers designed with Primer 5.0 software were shown in Supplementary Table S2. The gene expression was analyzed according to the method of Bu et al. [69 (link)]. Generally, total RNA from eight random fruit samples were isolated with RNAiso Plus (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Afterwards, RNA concentration was quantified by a Nanodrop spectrophotometer (NanoDrop 2000, Thermo-Fisher Scientific Inc., Bartlesville, OK, USA). The isolated RNA was reverse-transcribed to single-strand cDNA using a PrimeScript RT kit (TaKaRa, Shiga, Japan). qRT-PCR was performed using a SYBR^® Premix Ex TaqTM kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol on an ABI Step One RT-PCR System (Applied Biosystems, Beverly, MA, USA). The relative quantity of gene expression was analyzed using the comparative Ct method and calculated with 2^−ΔΔCt method [70 (link)]. Each measurement was performed with three replicates.

The stationary culture of ECOH having a starting OD₆₀₀ of 0.05 was inoculated in 30 ml of NB in 250 ml conical flasks containing NEs and incubated at 37 °C with shaking at 250 rpm for 24 h. The QIAzol Lysis Reagent (QIAGEN) was used to isolate total RNA as per the manufacturer’s protocol. Total RNA (1 μg) was used to synthesize cDNA using a PTC 200 thermocycler (MJ Research, Inc., Waltham, MA, USA). qRT-PCR was applied to examine the transcription levels of targeted genes. The reaction mixture contained 2 μL cDNA, 10 μL 2X SYBR master mix (Applied Biosystems, USA), 150 nM each primer, 0.4 μL reference dye, and RNA free water to make up 20 μL total volume. The analysis was carried out at 50 °C for 50 min followed by denaturation at 95 °C for 10 s, pursued by annealing at 55 °C for 10 s and extension at 60 °C for 35 s. To verify the absence of non-specific amplicons, a dissociation analysis was also performed. The relative fold expression change was determined using the CT method. Gene-specific primers were designed using Primer 3 (v 0.4.0) (Table S6). The rrsG gene was used as an internal control (housekeeping gene). ABI StepOne RT-PCR System (Applied Biosystems) was used for two independent cultures.

The total RNA was extracted from GSCLCs using the EasyPure RNA Kit (Transgen Biotech) following the manufacturer’s protocol, and the cDNA was synthesized using a TransScript Reverse Transcriptase kit (Transgen Biotech). Next, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using FastStart DNA Master SYBR Green (Roche Diagnostics, Indianapolis, IN, USA) on an ABI Step One RT-PCR system (Applied Biosystems, Foster City, CA, USA). Gene-specific primers were synthesized by GENEWIZ (Suzhou, China; Supplementary Table 2). The relative expression levels of TLRs, GDF15, LIF, and c-Fos were normalized to β-actin expression level and determined using the 2^−ΔΔCt method.

Total DNA was isolated from fecal samples using a Fast DNA Stool Mini Kit (QIAGEN). Quantification of DNA yield was performed using a Nanodrop 2000 spectrophotometer. The amplification of six bacteria species (Bacteroides spp., Bifidobacterium spp., Clostridium coccoides, Escherichia coli, Fusobacterium prausnitzii and Lactobacillus spp.) was conducted on an ABI StepOne RT‐PCR System (Applied Biosystems) using a QuantiTect SYBR Green RT‐PCR kit (Qiagen, 204245). Bacterial copy numbers were calculated according to the obtained cycle thresholds (CT). The primers for Real‐Time PCR detection are listed in the (Table S1).

Ten DEGs relevant to phenotype were selected to verify the RNA-seq data by the quantitative mRNA transcripts with real-time polymerase chain reaction (qRT-PCR) using the ABI Step One RT-PCR System (Applied Biosystems, CA) according to the manufacturer’s instructions. Total RNA samples were extracted using an RNA extraction kit (BioFlux, China). Then RNase-free DNase I was used to remove DNA contamination. Transcription of 500 ng RNA into cDNA was performed using a Revert Aid^TM First Strand cDNA synthesis kit (Fermentas, Shanghai, China) with random hexamer primers. Primers for detection of various genes are listed in Table 4. 16S rRNA gene was used as an internal control for quantification of relative gene expression. Each qRT-PCR reaction was conducted in a final volume of 50 μL. The thermal cycling profile was as follows: 94°C for 1 min, followed by 40 cycles of 94°C for 10 s, 55°C for 30 s, and 68°C for 15 s. Sterile water was used as negative control samples. The cycle threshold values (CT) were determined, and the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)) with 16S rRNA as the reference gene was used to calculate the relative fold differences. This experiment was repeated three times.

The RT-PCR reaction was performed using an ABI STEP ONE RT-PCR System (Applied Biosystems) in a 15-µl volume mixture using 2× FastStart Universal SYBR Green Master Mix (Bio-Rad, Hercules, CA, U.S.A.), which contained 0.375 μM primer and 0.5 μl cDNA. The protocol was followed as: 95°C denaturation for 3 min, 40 cycles of 95°C for 30 s, 60°C for 30 s, finally 72°C for 1 min. Relative gene expression was quantified using the comparative ΔC_T method. The relative expression was normalized to GAPDH mRNA expression. Primer sequences were listed in Table 1.