Table 1.
Primer sequences and references
| Primer name | Gene product | Sequence | Product length (bp) | NCBI reference sequence no.a | % PCR efficiencyb |
|---|---|---|---|---|---|
| Bov IDO | IDO | 5′-CGAATATACTTGTCTGGTTGG (forward) | 139 | NM_001101866 | 99.3 |
| 5′-GGAGAACATCAAAGCACTG (reverse) | XM_001490681.1 | ||||
| Bov RGc | ZC3H13 | 5′-ATGCTTCAGACGGGATTCTG (forward) | 204 | XM_002691780 | 98.2 |
| 5′-TGTTGGATACCGGACTGTGA (reverse) | |||||
| Hu βglucd | β-Glucuronidase | 5′-TGTTTGAACAGCTACTACTCTTG (forward) | 131 | NM_000181.3 | 101.8 |
| 5′-GCTCCATACTCGCTCTGAAT (reverse) | |||||
| Hu GAPDH | GAPDH | 5′-CATCAAGAAGGTGGTGAAGCAG (forward) | 119 | NM_002046 | 92.4 |
| 5′-CGTCAAAGGTGGAGGAGTTGG (reverse) | |||||
| Hu Hist | Histone H3 | 5′-GTAAAGCACCCAGGAAGCAA (forward) | 137 | NM_002107.3 | 96.1 |
| 5′-AGTGGACTTCTGATAACGTCTAAT (reverse) | |||||
| Hu IDO | IDO | 5′-AGTCCGTGAGTTTGTCCTTTC (forward) | 145 | NM_002164 | 95.9 |
| 5′-CTTTGGCTGCTGGCTTGC (reverse) | |||||
| Hu IL-12B | IL-12B (p40) | 5′-GCAAGAGCAAGAGAGAAAAGAAAG (forward) | 135 | NM_002187.2 | 105.4 |
| 5′-ATGCCCATTCGCTCCAAGAT (reverse) | |||||
| Hu IL-10 | IL-10 | 5′-CAGCAGAGTGAAGACTTTCTTTC (forward) | 143 | NM_000572.2 | 103.8 |
| 5′-GCATCACCTCCTCCAGGTAA (reverse) |
a
NCBI nucleotide reference sequence accession number.
b
Efficiency of the primer set in qPCR, determined from a cDNA standard curve.
c
Bov RG, reference gene for bovine PBMC (identified by microarray studies) encoding predicted Bos taurus zinc finger CCCH-type domain-containing protein 13 (ZC3H13; Affymetrix identifier Bt.28167.1.S1).
d
Human β-glucuronidase was selected as the reference gene for THP-1 studies, as it had the lowest standard deviations (SD) (0.18) across treatment groups (control and M. avium subsp. paratuberculosis infected) and time points (0, 4, 24, and 48 h) at levels closest to those of the genes of interest (48).