Quantstudio 3 real time pcr system

The RT-qPCR was carried out following the protocol of SYBR Green Dye kit (Vazyme, Nanjing, China). The reaction system (20 μL) included 1.3 μL of cDNA, 1 μL of forward primers, 1 μL of reverse primers, 6.7 μL of DEPC water, and 10 μL of SYBR Green Dye. RT-qPCR was conducted on an Applied Biosystems QuantStudio 3 system (Thermo Fisher, Waltham, MA, USA) following the conditions: pre-denaturation step at 95 °C for 5 min, 40 amplification cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 15 s, followed by a final elongation step at 72 °C for 10 min. The reaction was performed using an Applied Biosystems QuantStudio 3 Real-Time PCR System (Themo Fisher). All the reactions were performed in triplicate. The relative expression of piRNA was calculated using the 2^−ΔΔCt method [28 (link)]. Detailed information about the primers that were used in this work is shown in Table S1.

To validate sequencing results, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to measure the levels of key genes found to be DE. Total RNA was converted into cDNA as previously described (11 (link)). Expression levels of the genes C-X-C motif chemokine ligand 2 (CXCL2), phosphoenolpyruvate carboxykinase 1 (PCK1), malic enzyme 1 (ME1), dipeptidase 1 (DPEP1), and stearoyl-CoA desaturase (SCD) were quantified using TATA-box binding protein (TBP) as the housekeeping gene (Supplementary Table 2). All qPCR assays were done in triplicate using qPCRBIO SyGreen Blue Mix Lo-ROX (PCRBIOSYSTEMS) and the QuantStudio3 Real-Time PCR System (Themo Fisher). For the standard curve, cDNA from each sample of the groups/sections was pooled at equal concentrations. The analysis of fold change expression was done by the delta-delta Ct method with Wilcoxon and t-testing for significance.

Total RNA was extracted from DP cells using TRIzol™ Reagent (Invitrogen; Thermo Fisher Scientific), and cDNA was synthesized using reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific). For real-time PCR, QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) was used. Total reaction volume of 20 μL contained 1-μL cDNA, 500 nM of each primer, and 10-μL SensiFAST™ SYBR Lo-ROX Kit (Bioline, Taunton, MA, USA). Samples were analyzed in triplicate, and β-actin was used as an internal control to normalize the relative quantity of gene expression. For PCR, total volume of 50 μL contained 1-μL cDNA, 200 nM of each primer, Dream Taq DNA Polymerase and Dream Taq buffer (Thermo Fisher Scientific) for 35 cycles. The following primer pairs were used: Wnt3a, 5′-AGTGCAAATGCCACGGACTA-3′ (forward), 5′-TTGGGCTCGCAGAAGTTAGG-3′ (reverse); Wnt7a, 5′-CAGAATGCCCGAACCCTCAT-3′ (forward), 5′-TAGCCT GAGGGGCTGTCTTA-3′ (reverse); β-catenin, 5′-CCATCACCACGCTGCATAAT-3′ (forward), 5′-GAGCAGACAGACAGCACCTT-3′ (reverse); Lef1, 5′-GGCATC CCTCATCCAGCAAT-3′ (forward), 5′-GTTGATAGCTGCGCTCTCCT-3′ (reverse); Gsk3b, 5′-AGAACCACCTCCTTTGCGGA-3′ (forward), 5′-GTGGTTACCTTGCTGCCATCT-3′ (reverse); Fzd7, 5′-GGTGGATGGTGACCTACTCA-3′ (forward), 5′-GCTCGTAAAAGTAGCACGCC-3′ (reverse); and β-actin, 5′-AAGATCCTGACCGA GCGTGG-3′ (forward), 5′-CCGCTCATTGCCGATAGTG-3′ (reverse).

RNA was purified by using TRI Reagent (Sigma-Aldrich) and 3 μg was retro-transcripted using MMLV (Promega, Madison, WI, USA). qPCR was performed in triplicate using validated qPCR primers (BLAST), Applied Biosystems™ Power™ SYBR™ Green Master Mix and the QuantStudio3 Real-Time PCR System (Thermo Fisher Scientific, Whaltam, MA, USA). mRNA levels were normalized to RPL8 mRNA and quantified through the 2^−ΔΔCt method. The list of primers is reported in Table 1.

Bacterial translocation was determined as bacterial load in liver tissue and was quantified by real-time PCR using the 16S rRNA gene consensus sequence as described by Banerjee et al. [47 (link)]. The total load of bacteria in the liver was determined using primer sequences to amplify the highly conserved sequence for a broad species consensus. Livers were removed aseptically and stored at −80 °C until use. Bacterial translocation was quantified by real-time PCR. Briefly, liver tissue was homogenized in a MP FastPrep 24 Instrument (MP biomedicals, Irvine, CA) using Green Bead Lysis kits (Next Advance, Troy, NY). DNA was isolated from liver lysates using a DNA purification kit (Promega, Madison, WI). Bacterial DNA and liver genomic DNA concentration were quantified using Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). Serially diluted bacterial genomic DNA was used to generate the standard curve. Real-time PCR was performed using PowerUp SYBR green PCR master mix (Applied Biosystems, Foster City, CA) and 16S rRNA gene targeted primers, forward (5′-ACTCCTACGGGAGGCAGCAGT-3′) and reverse (5′-TATTACCGCGGCTGCTGGC-3′) in a QuantStudio 3 Realtime PCR System (Thermo Fisher Scientific, Waltham, MA). PCR-derived bacterial counts were expressed as nanogram bacterial DNA per gram mouse liver tissue.