Open array plate

A custom Open-Array plate (Applied Biosystems, Foster City, CA, USA) was designed with a total of 56 selected genes related to intestinal health (Supplementary Table S4). Details regarding genes and primers can be found in previous published work⁵⁵ . Multiplex real-time qPCRs were performed in a QuantStudio 12 K Flex Real-Time PCR system (ThermoFisher Scientific, Waltham, MA) using TaqMan Open-Array Real-Time PCR Custom Assays. A final cDNA volume of 6 μl from each sample was transferred to 384-well plates and analysed per duplicate. One sample was used as an inter-plate control to check the replication of results from different plates.
Gene expression data analysis was performed as specified by Reyes-Camacho et al⁵⁵ .

The expression of a total of 52 genes related to intestinal health was studied through a custom OpenArray plate (Applied Biosystems, Foster City, CA, USA). Full details of the method has been previously described by González-Solé et al^{81 (link)}. The method had been optimized to quantify the expression of relevant genes in jejunum. Genes were selected based on previous knowledge and other reported works in the literature. GAPDH, ACTB, TBP and B2M were used as housekeeping genes. RNA was extracted from 100 mg of frozen jejunum tissue with the RiboPure kit (Ambion, Foster City, CA, USA) following the manufacturer’s protocol. A final cDNA volume of 6 μl from each sample was transferred per duplicate to a TaqMan OpenArray custom plate for gene expression and run in a QuantStudio 12 K Flex Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Gene expression data analysis was performed as specified by González-Solé et al^{81 (link)}. A list of the 52 genes included in the custom plate can be found in Supplementary information (Supplementary Table S1).