384 well real time pcr system

The NE-induced host immune response of inflammatory gene expression was quantified in ileal tissue samples. The total RNA was extracted by the TRIzol method from ileal tissue samples of birds at day 25 as described before [1 (link),3 (link)] and cDNA was prepared using M-MLV (NE Biolabs). The accumulation of the proinflammatory genes of Ifnγ, Mmp9, and Gapdh in the ileum tissue was determined using SYBR Green PCR Master mix (Bio-Rad) on a Bio-Rad 384-well Real-Time PCR System as described before [1 (link)]. The gene expression of the fold-change was calculated using the ΔΔCt method [1 (link),3 (link)] and Gapdh as an internal control.
The degree of ileal cells apoptosis in the intestinal tissue was assessed using the TUNEL assay as described before [1 (link),3 (link)]. The TUNEL assay detected the late phase of cellular apoptosis based on the fragmentation of nucleic acids. In brief, ileal tissue slides were deparaffinized three times with xylene wash and then rehydrated with 100%, 95%, and 70% ethanol. The tissues were then incubated at 37 °C for 90 min with the TUNEL solution (5 μM Fluorescein-12-dUTP (Enzo Life Sciences), 10 μM dATP, 1 mM pH 7.6 Tris-HCl, 0.1 mM EDTA, 1U TdT enzyme (Promega)). For nucleus visualization, the slides were counter-stained utilizing DAPI. Using a Nikon TS2 fluorescent microscopy, the fluorescent green apoptotic cells were examined and imaged.

mRNA levels of proinflammatory genes of mouse Il1β, Tnfα, and Cxcl2, and chicken Ifnγ, Il1β, and Il8-1, were determined using the SYBR Green PCR Master mix (Bio-Rad Hercules, CA, USA) on a Bio-Rad 384-well Real-Time PCR System and normalized to the respective Gapdh. The primer sequences were reported before [10 (link),14 (link),42 (link)] and are in Supplementary Table S1. The PCR reactions were performed according to the manufacturer’s recommendation. Similarly, the level of C. perfringens in the intestinal luminal and tissue was also quantified by RT-PCR targeting the bacterial 16S rDNA, as described before [14 (link)].

Cecal digesta samples were collected and DNA was extracted using bead beater disruption and phenol: chloroform separation as describe before [34 (link)]. The levels of five phylum bacteria were determined using SYBR Green PCR Master mix (Bio-Rad) on a Bio-Rad 384-well Real-Time PCR System. The PCR reactions were performed according to the manufacturer’s recommendation. The following gene primers [34 (link), 36 (link)] were used: Universal 16S: 16s357F: 5’-CTCCTACGGGGAGGCAGCAA-3’, 16s1392R: 5’-ACGGGCGGTGTGTRC-3’; α-proteobacteria: α682F 5’-CIAGTGTAGAGGTGAAATT-3’, 908αR 5’-CCCCGTCAATTCCTTTGAGTT-3’; γ-proteobacteria: 1080γF 5’-TCGTCAGCTCGTGTYGTGA-3’ γ1202R 5’-CGTAAGGGCCATGATG-3’; Bacteroidetes: 798cfbF 5’-5’-CRAACAGGATTAGATACCCT-3’ cfb967R 5’-GGTAAGGTTCCTCGCGTAT-3’; Firmicutes: 928F-Firm 5’-TGAAACTYAAAGGAATTGACG-3’ 1040 Firm R 5’-ACCATGCACCACCTGTC-3’; Actinobacteria: Act920F3 5’-TACGGCCGCAAGGCTA-3’ Act1200R 5’-TCRTCCCCACCTTCCTCCG-3’. The relative fold change of each phylum in one sample were normalized to universal 16S. The percentage of each phylum was then calculated as the phylum relative folds divided by total folds of all five phyla.