Plasmid dna

Absolute quantitative real-time PCR (qPCR) was performed to determine the 16S copy number of total bacterial species as well as the copy number of Lactobacillus spp., Bifidobacterium spp., and the Escherichia subgroup. The primers were previously validated (Supplementary Table S2), and qPCR was performed using a CFX96 Real-time System (Bio-Rad Laboratories, Inc., Hercules, CA, United States). The PCR conditions were: one cycle at 95°C for 3 min, 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 10 s, and one cycle at 72°C for 5 min. We verified the efficiency of the primers and calculated the absolute abundances of bacteria based on standard curves. Standard curves were prepared using plasmid DNA (TransGen Biotech, Beijing, China) containing each unique 16S rDNA insert. The absolute abundances are expressed as log₁₀ of copy number per gram of ileal digesta.

Total RNA was extracted using Trizol reagent (TransGen) and reverse-transcribed using the Transcript First-Strand cDNA Synthesis SuperMix Kit (TransGen). mRNA levels were quantified by real-time PCR. Primers were designed using Primer 5.0 software (PREMIER Biosoft International, Palo Alto, CA, United States) (Supplementary Table S3). Real-time PCR was performed in a CFX96 Real-time System. The PCR conditions consisted of one cycle at 95°C for 3 min, 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 10 s, and one cycle at 72°C for 5 min. We verified the specificity of primers using melting curves and the fragment size of the amplification products, and verified the efficiency using standard curves prepared using plasmid DNA (TransGen) containing each gene sequence insert. We compared the stabilities and efficiencies of four candidate housekeeping genes (GAPDH, PGK1, 18S rRNA, and β-actin) in the lamb intestine and selected β-actin as the internal control (Ma et al., 2015a ). The 2^-ΔΔCT method was used to analyze the data (Livak and Schmittgen, 2001 (link)).