E-MTAB-5606
RNA-seq of porcine peripheral whole blood from pig divergently selected for residual feed intake and treated with lipopolysaccharide
Yorkshire pigs were divergently selected for residual feed intake (RFI) for 8 generations. Pigs with low RFI have reduced feed intake. Based on the resource allocation theory, we hypothesize that these low RFI pigs might have compromised immune response to immune stimulation. To test this hypothesis, twenty eight gilts of initial body weight (BW) of 63 ± 4 kg from pigs selected for low RFI and high RFI were randomly selected for the ISU RFI selection project and utilized in two replicates (14 pigs/replicate) for this study. All pigs were housed in individual metabolism pens, had free access to water and were fed a corn-soy-based diet twice daily, and feed restricted (1.5 kg/day) as previously described. After a 9-day adaptation period pigs were randomly assigned within line to either a control (n = 6, three pigs per line) or lipopolysaccharide (LPS) challenge (n = 8, 4 pigs per line) group. Pigs in the challenge group were then repeatedly challenged with an intramuscular injection of 30, 36, 39, and 42 µg/kg BW of LPS from E. coli O5:B5 (sigma-Aldrich, St. Louis, MO) dissolved in saline solution at time points 0, 48, 96, and 144 hours post initial injection (hpi) to compensate for LPS tolerance after initial injection, respectively, while animals in the control group were injected with an equivalent volume of saline solution at the equivalent time points. Rectal temperatures of individual pigs were measured immediately before the initial injection (serving as baseline, called “0 hpi” for convenience), and 2, 4, 6, and 24 hpi, and 0, 4, 24 hours after each subsequent injection for all pigs. Blood samples were collected from the jugular vein into TempusTM Blood RNA tubes (Life Technologies, Grand Island, NY) for long-term storage at -80 °C, EDTA tubes (BD, Franklin Lakes, NJ) for CBC tests and serum tubes for cytokine assays at 0, 2, 6, 24 and 168 hpi. At 168 hpi, all pigs were euthanized via barbiturate overdose, exsanguinated and tissue samples including the longissimus dorsi muscle, liver, spleen, and ileum were isolated, cleaned an frozen for later use. This study only focused on samples and data collected for the first 24 hours. Total RNA was extracted from the 32 blood samples of the treatment group in Replicate 2, collected at 0, 2, 6, and 24 hpi, by using preserved blood RNA purification kit I (Norgen Biotek Corp, Thorold, Ontario) per manufacturer’s instruction. On-column DNA digestion was performed as described using DNase I (Qiagen, Valencia, CA). Globin transcripts (HBB and HBA) were depleted by following an RNase H-mediated method. The quantity and integrity of the RNA were monitored by using Nanodrop 2000 (Thermo Scientific, Waltham, MA) and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) before and after globin depletion. The efficiency of globin depletion of each sample was checked by conventional RT-qPCR with ß-actin and GAPDH as the internal controls. Globin depletion efficiencies for all RNA samples were above 85%.
Yorkshire pigs were divergently selected for residual feed intake (RFI) for 8 generations. Pigs with low RFI have reduced feed intake. Based on the resource allocation theory, we hypothesize that these low RFI pigs might have compromised immune response to immune stimulation. To test this hypothesis, twenty eight gilts of initial body weight (BW) of 63 ± 4 kg from pigs selected for low RFI and high RFI were randomly selected for the ISU RFI selection project and utilized in two replicates (14 pigs/replicate) for this study. All pigs were housed in individual metabolism pens, had free access to water and were fed a corn-soy-based diet twice daily, and feed restricted (1.5 kg/day) as previously described. After a 9-day adaptation period pigs were randomly assigned within line to either a control (n = 6, three pigs per line) or lipopolysaccharide (LPS) challenge (n = 8, 4 pigs per line) group. Pigs in the challenge group were then repeatedly challenged with an intramuscular injection of 30, 36, 39, and 42 µg/kg BW of LPS from E. coli O5:B5 (sigma-Aldrich, St. Louis, MO) dissolved in saline solution at time points 0, 48, 96, and 144 hours post initial injection (hpi) to compensate for LPS tolerance after initial injection, respectively, while animals in the control group were injected with an equivalent volume of saline solution at the equivalent time points. Rectal temperatures of individual pigs were measured immediately before the initial injection (serving as baseline, called “0 hpi” for convenience), and 2, 4, 6, and 24 hpi, and 0, 4, 24 hours after each subsequent injection for all pigs. Blood samples were collected from the jugular vein into TempusTM Blood RNA tubes (Life Technologies, Grand Island, NY) for long-term storage at -80 °C, EDTA tubes (BD, Franklin Lakes, NJ) for CBC tests and serum tubes for cytokine assays at 0, 2, 6, 24 and 168 hpi. At 168 hpi, all pigs were euthanized via barbiturate overdose, exsanguinated and tissue samples including the longissimus dorsi muscle, liver, spleen, and ileum were isolated, cleaned an frozen for later use. This study only focused on samples and data collected for the first 24 hours. Total RNA was extracted from the 32 blood samples of the treatment group in Replicate 2, collected at 0, 2, 6, and 24 hpi, by using preserved blood RNA purification kit I (Norgen Biotek Corp, Thorold, Ontario) per manufacturer’s instruction. On-column DNA digestion was performed as described using DNase I (Qiagen, Valencia, CA). Globin transcripts (HBB and HBA) were depleted by following an RNase H-mediated method. The quantity and integrity of the RNA were monitored by using Nanodrop 2000 (Thermo Scientific, Waltham, MA) and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) before and after globin depletion. The efficiency of globin depletion of each sample was checked by conventional RT-qPCR with ß-actin and GAPDH as the internal controls. Globin depletion efficiencies for all RNA samples were above 85%.