Piglet Jejunal Mucosal RNA Extraction and qRT-PCR Analysis

Total RNA was extracted from the piglet jejunal mucosal samples using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. DNase treatment was performed to remove contaminating DNA using the TURBO DNA-free™ DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the recommended protocol. The quantity and quality of the RNA were evaluated using a Nanodrop ND 1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA) and agarose gel electrophoresis, respectively. A total of 1000 ng of RNA was then converted into complementary DNA using the High-Capacity RNA-to-cDNA™ Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Duplex Real Time PCR reactions contained 2 µL cDNA and 8 µL mix containing primers, probe (Additional file 1: Table S1) and 2X TaqMan Mastermix, and were run in triplicate on the Applied Biosystems QuantStudio™ 7 Flex Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with the following thermocycler settings: 50 °C for 2 min, 95 °C for 2 min and 40 cycles of 95 °C for 1 s and 60 °C for 20 s. Hydroxymethylbilane synthase (HMBS) was used as housekeeping gene. The following genes were selected and analysed: innate immune signal transduction adaptor (MyD88), nuclear factor kappa B subunit 2 (NFKB2), Occludin (OCLN), Tight junction protein 1 (ZO-1), Mucin 13 cell surface associated (MUC13), glutathione peroxidase 2 (GPX-2), Claudin-4 (CLAUD4), Claudin-3 (CLAUD3), polymeric immunoglobulin receptor (PIGR), branched chain amino acid transaminase 2 (BCAT2), ornithine decarboxylase 1 (ODC1), solute carrier family 6 (neutral amino acid transporter), member 19 (SLC6A19), solute carrier family 7 member 9 (SLC7A9), solute carrier family 1 member 5 (SLC1A5), solute carrier family 38 member 2 (SLC38A2).
QuantStudio Design and Analysis Software v2.5 (Thermo Fisher Scientific, Waltham, MA, USA) was used for determining the gene expression cycle threshold (Ct) values. For each sample the Ct value of the HMBS gene was subtracted from the Ct value of the target gene (ΔCt). The average ΔCt value of the reference animals was then subtracted from the ΔCt value of all the samples (ΔΔCt). The expression of the target gene was given as fold change calculated by 2^−ΔΔCt.