Table 3.
Panel of target genes evaluated in the ileum and colon
| Target gene | Accession No. | Forward primer 5’ to 3′ Reverse primer 5′ to 3’ |
Amplicon length, bp | Tm, °C |
|---|---|---|---|---|
| Immune response | ||||
| IL1A | NM_214029.1 |
F: CAGCCAACGGGAAGATTCTG R: ATGGCTTCCAGGTCGTCAT |
76 |
63.0 60.5 |
| IL6 | NM_214399.1 |
F: GACAAAGCCACCACCCCTAA R: CTCGTTCTGTGACTGCAGCTTATC |
69 |
59.8 62.7 |
| IL7 | NM_214135.2 |
F: GAGTGACTATGGGCGGTGAGA R: GCGGGCGTGGTCATGA |
63 |
61.8 56.9 |
| CXCL8 | NM_213867.1 |
F: TGCACTTACTCTTGCCAGAACTG R: CAAACTGGCTGTTGCCTTCTT |
82 |
61.9 61.7 |
| IL10 | NM_214041.1 |
F: GCCTTCGGCCCAGTGAA R: AGAGACCCGGTCAGCAACAA |
71 |
63.4 63.1 |
| IL17A | NM_001005729.1 |
F: CCCTGTCACTGCTGCTTCTG R: TCATGATTCCCGCCTTCAC |
57 |
60.6 60.4 |
| IL22 | XM_001926156.1 |
F: GATGAGAGAGCGCTGCTACCTGG R: GAAGGACGCCACCTCCTGCATGT |
112 |
66.0 66.0 |
| IFNG | NM_213948.1 |
F: TCTAACCTAAGAAAGCGGAAGAGAA R: TTGCAGGCAGGATGACAATTA |
81 |
61.1 61.5 |
| TNF | NM_214022.1 |
F: TGGCCCCTTGAGCATCA R: CGGGCTTATCTGAGGTTTGAGA |
68 |
62.5 62.8 |
| TGFB1 | NM_214015.1 |
F: AGGGCTACCATGCCAATTTCT R: CGGGTTGTGCTGGTTGTACA |
101 |
60.6 61.7 |
| FOXP3 | NM_001128438.1 |
F: GTGGTGCAGTCTCTGGAACAAC R: AGGTGGGCCTGCATAGCA |
68 |
60.6 61.2 |
| CCL20a | NM_001024589.1 |
F: GCTCCTGGCTGCTTTGATG R: TTGCTTGCTGCTTCTGACTTG |
66 |
58.8 57.9 |
| TLR4 | NM_001293317.1 |
F: TGCATGGAGCTGAATTTCTACAA R: GATAAATCCAGCACCTGCAGTTC |
140 |
57·1 60·6 |
| TP53a | NM_213824.3 |
F: CCGGGTGGAAGGGAATTT R: CCACAACGCTGTGTCGAAAA |
68 |
56.0 57.3 |
| STAT3a | NM_001044580 |
F: TCTTGAGAAGCCAATGGAGATTG R: TGGAGGAGGCGGGACTCT |
69 |
58.9 60.5 |
| Intestinal integrity | ||||
| MUC2 | AK231524 |
F: CAACGGCCTCTCCTTCTCTGT R: GCCACACTGGCCCTTTGT |
70 |
63.1 62.1 |
| TJP1/ZO-1 | XM_005659811.1 |
F: TGAGAGCCAACCATGTCTTGAA R: CTCAGACCCGGCTCTCTGTCT |
76 |
59.9 60.0 |
| Cholinergic receptor | ||||
| CHRM1a | NM_214034.1 |
F: GCCATGGCCGCCTTCT R: GGTTCTCTGTCTCCCGGTAGATG |
76 |
56.9 64.2 |
| NADPH oxidases | ||||
| NOX1a | XM_003484140.3 |
F: CTTTGAAAGGATCCTCCGATTTT R: ATGGATACATGACCACCTTGGTAA |
71 |
57.1 59.3 |
| DUOX2a | NM_213999.2 |
F: CTGGGCCTTGACATAGATGAGAT R: GGCAAAAAGGTGTCTGAAGAAGA |
108 |
60.6 58.9 |
| Reference genes | ||||
| PPIA | NM_214353.1 |
F: CGGGTCCTGGCATCTTGT R: TGGCAGTGCAAATGAAAAACT |
75 |
62.1 60.7 |
| B2M | NM_213978.1 |
F: CGGAAAGCCAAATTACCTGAAC R: TCTCCCCGTTTTTCAGCAAAT |
83 |
58.2 58.4 |
| GAPDH | AF017079.1 |
F: CAGCAATGCCTCCTGTACCA R: ACGATGCCGAAGTTGTCATG |
72 |
62.2 62.1 |
bp, Base pairs; Tm, Melting temperature; IL1A, Interleukin 1 alpha; IL6, Interleukin 6; IL7, Interleukin 7; CXCL8, C-X-C motif chemokine ligand 8; IL10, Interleukin 10; IL17A, Interleukin 17 alpha; IL22, Interleukin 22; IFNG, Interferon gamma; TNF, Tumour necrosis factor; TGFB1, Transforming growth factor beta 1; FOXP3, Forkhead box P3; CCL20, C-C motif chemokine ligand 20; TLR4, Toll-like receptor 4; TP53, Tumour protein p53; STAT3, Signal transducer and activator of transcription 3; MUC2, Mucin 2; TJP1/ZO-1, Tight junction protein 1/Zona occludens 1; CHRM1, Cholinergic receptor muscarinic 1; NOX1, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1; DUOX2, Dual oxidase 2; PPIA, Peptidylprolyl isomerase A; B2M, Beta-2-microglobulin; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase
aGenes encoding proteins with an established role in facilitating or inhibiting Salmonella infection. These proteins are associated with chemotaxis (CCL20), production of reactive oxygen species (NOX1, DUOX2) anti-inflammatory activity (STAT3, CHRM1) and cell survival and death (TP53) [54–59]