Sybr green technology

EL4 cells (Sigma-Aldrich) were grown in DMEM (Gibco). At 24 h after the cells were seeded onto a 12-well plate, the cells were treated with ligands 1–3 (from 10 mM stock in DMSO) or DMSO. After 24 h, the cells were collected and RNA was isolated using a RNeasy Plus Micro Kit (Qiagen) and reverse transcribed using the iScrip cDNA biosynthesis kit (Bio-Rad). Quantitative RT–PCR was performed to analyse mRNA levels of mouse IL17a levels using SYBR green technology (Bio-Rad) on a CFX Real-Time System (Bio-Rad). Primer sequences used for IL17a:45 (link) Fw: 5′-ctccagaaggccctcagactac-3′, Rev: 5′-ctgtgtcaatgcggagggaaagct-3′ and Gapdh: Fw: 5′-ggtggacctcatggcctaca-3′, Rev: 5′-ctctcttgctcagtgtccttgct-3′. The level of IL17a mRNA expression was normalized to that of Gapdh expression. All data are expressed as the mean±s.e.m. (n=6). Statistical analysis was performed using an one-way analysis of variance comparing against the DMSO control following Dunnett post hoc test.

Total RNA was extracted from mouse hearts using the TRIzol reagent (Invitrogen, CA, USA). The mRNA expression levels of monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), and inducible nitric oxide synthase (iNOS) were determined using SYBR Green technology (Bio-Rad, CA, USA) with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. Quantitative values were obtained from the threshold cycle value (Ct), and the 2^-△△Ct method was used to determine the relative gene expression levels. The primers were as follows: MCP-1, forward, 5′-TCTGGGCCTGCTGTTCACA-3′, reverse, 5′-GGATCATCTTGCTGGTGAATGA-3′; ICAM-1, forward, 5′-GCCTTGGTAGAGGTGACTGAG-3′, reverse, 5′-GACCGGAGCTGAAAAGTTGTA-3′; iNOS, forward, 5′-GTTCTCAGCCCAACAATACAAGA-3′, reverse, 5′-GTGGACGGGTCGATGTCAC-3′; and GAPDH, forward, 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse, 5′-TGTAGACCATGTAGTTGAGGTCA-3′.

For each sample, 1 μg of RNA was used to generate cDNA using qScript cDNA SuperMix kit (Quantabio cat# 95048-100). In quantitative PCR, aliquots of cDNA samples with three replicates were used to detect differential expression of Fabp2, Krt20, Car1, Muc2, Ephb2, Lgr5, Ascl2 with SYBR Green technology (Bio-Rad cat# 1725124), while β-actin was applied as an endogenous control. These marker genes were used as previous studies have reported that they represent markers of stem cell or differentiated cells in the colon epithelium. The markers we used for stem cells are Ephb2, Lgr5, and Ascl2, which have been shown to be expressed at high levels in intestinal stem cells^{37 (link),55 (link)}. The differentiated cell markers used are: (a) Fabp2, which is an intestinal fatty acid-binding apoprotein expressed in intestinal enterocytes^{56 (link)–58 (link)}; (b) Car1 (Carbonic anhydrase I) is expressed in colonocyte^{56 (link),57 (link)}; (c) Krt20 (Cytokeratin 20) is expressed in differentiated epithelial cells^{55 (link),59 (link)}; (d) Muc2 (mucin) is expressed in goblet cells^{56 (link),60 (link)}. The delta-delta Ct method and log2-fold change were used to calculate the relative gene expression. The exact primer sequences are listed in Supplementary Table 4. The experiment was repeated three times.

Total RNA was isolated from cells after various treatments and transfections as described in the figure legends for each specified experiment using the RNeasy mini kit (Qiagen, Beverly, MA, USA) according to the standard protocol provided by the manufacturer, with on-column DNA digestion. RNA integrity and concentration were analyzed using Bioanalyzer, and 100 ng of RNA was retrotranscribed into cDNA using the First Strand cDNA synthesis kit from Roche Applied Science. SYBR Green technology (Bio-Rad, Thermo Fisher, Waltham, MA, USA) was used for all real-time PCR experiments. Amplification was done with the real-time PCR analyzer (Bio-Rad cfx96). The PCR mixture (25 µL) contained 12.5 µL of 2 SYBR Green PCR Master Mix (Bio-Rad), 5 µL of diluted RT product (1:20), and 0.5 µM sense and antisense primer sets. The real-time PCR assays were done in three individual experiments with duplicate samples using standard conditions in a CFX96 real-time PCR detection machine. After incubations at 95 °C for 3 min, the amplification protocol consisted of 50 cycles of denaturing at 95 °C for 10 s, annealing, and extension at 60 °C for 30 s. The standard curve was made from a series dilution of template cDNA. Expression levels of tested genes were calculated after normalization with the housekeeping gene GAPDH. Primer sequences used in QPCR are presented in Table 2.

Tissues were collected at sacrifice and snap frozen in liquid nitrogen to avoid tissue degradation. Liver, ileum and proximal colon tissues were ground into powders and 30–60 mg of tissues were used for RNA extraction using RNeasy Kit from Qiagen. cDNA synthesis was performed on 1 µg of RNA using High-Capacity cDNA Reverse Transcriptase kit (Applied biosystems). qPCR analyses were performed using the SYBR Green technology (Bio-Rad) and primer sequences are listed in online supplemental table 2.

Real-time PCR was performed to determine the gene expression of MIF, CD74, MMP-9, MCP-1, TNF-α and Jun activation domain-binding protein 1 (Jab1) in atherosclerotic lesions by using SYBR Green Technology (Bio-Rad, California, USA), and the mouse housekeeping gene β-actin was applied as an internal control. The sequences of primers for β-actin or target gene sequence were mentioned on Table S2. The data were analysed by the 2^−ΔΔCT method. All experiments were repeated for at least three times.