Taqman gene assays

Quantitative RT-PCR (qRT-PCR) was carried out by a Light Cycler 480 sequence detection system (Roche Diagnostics) on total RNAs (100 ng) reversely transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems), according to the manufacturer’s instructions. Each cDNA sample was run in duplicate for either each target (VEGFA and CCNA1) or GAPDH endogenous control. TaqMan gene assays, supplied as ready solutions, and TaqMan Universal PCR Master Mix were provided by Applied Biosystems. Quantification of qRT-PCR signals was performed using the (2^{-Delta DeltaCt}) method [20 (link)], which calculates relative changes in gene expression of the target gene normalized to the GAPDH endogenous control and relative to a calibrator sample. The values obtained were represented as relative quantity of mRNA level variations.

To evaluate expression of different genes of interest, mLN and 2 × 0.5 cm pieces of SI, harvested 27 and 37 cm distal from the stomach were collected and stored in RNAlater (Invitrogen, Carlsbad, CA, USA) at −20 °C. RNA extraction, cDNA synthesis, and RT‐qPCR were performed as previously described.^[43] For RNA extraction from mLN, QIAzol Lysis Reagent (79306, Qiagen, Hilden, Germany) and RNeasy Lipid Tissue Mini Kit (74804, Qiagen) were used in accordance with manufacturers’ protocol. Taqman gene assays (Applied Biosystems, Thermo Fisher Scientific, MA, USA) used were: Ocln (occludin Rn00580064_m1), TSLP (thymic stromal lymphopoietin, Rn01761072_m1), IL‐1β (interleukin 1beta, Rn00580432_m1), IL‐4 (interleukin 4, Rn01456866_m1), IL‐10 (interleukin 10, Rn01483989_m1), INF‐γ (interferon gamma, Rn00594078_m1), TGF‐β (transforming growth factor beta, Rn005720_m1), FoxP3 (forkhead box P3, Rn01525092_m1), and CX3XR1 (C‐X3‐C chemokine receptor 1, Rn00591798_m1). The levels of different gene expression were shown as the relative gene expression by means of 2^−deltaCT method using B2m (Beta‐2‐microglobulin Rn00560865_m1) and Sdha (Succinate dehydrogenase complex Rn00590475_m1) as normalization genes.

Total RNA was extracted from each sample treated with UVB using TRIzol reagent (Invitrogen, Carlsbad, CA). Real-time PCR was performed using TaqMan Gene assays (Applied Biosystems, Foster City, CA) specific for the genes encoding MMP-1, MMP-9, tissue inhibitor of metalloproteinase (TIMP), involucrin, filaggrin, loricrin, IL-1β, IL-6, IL-8, on a QuantStudio^TM 6 Flex Real-Time PCR systems. Relative amounts of cDNA were calculated by the relative quantification (ΔΔCt) method. Each sample was run in triplicate and the gene encoding β-actin was used as a control to normalize for differences in the amount of total RNA in each sample.

Total mRNA isolated from cells or frozen tissues using Qiagen kits (Valencia, CA) was reverse transcribed to generate cDNA. Real-time PCR was performed in triplicate on the generated cDNA using Taqman^® gene assays (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions [13 (link)]. For murine Cd31, Vegfr2, VE-cadherin (Cdh5), and Gapdh, the Mm00476702_m1, Mm01222421_m1, Mm00486938_m1, and Mm99999915_g1 Taqman primer sets and Taqman assay reagent were used. The real-time PCR results were normalized to Gapdh.