gender: male age: 3 to 6 weeks genetic background: outbred tissue: distal ileum
Treatment protocol
The bovine ligated ileal loop surgical procedure has been described. Briefly, the calves were fasted for 12 hours prior to the surgery. Anesthesia was induced with propofol (Abbot Laboratories, Chicago, IL) followed by placement of an endotracheal tube and maintainance with isofluorane (Isoflo, Abbot Laboratories) for the duration of the experiment. A laparotomy was performed, the distal ileum was exposed, and 21 loops approximately 6 to 9 cm in length were ligated with umbilical tape, leaving 1 cm loops between them. Overnight bacterial cultures were grown as described above. The ileal loops were inoculated by intralumenal injection of 3 ml of a suspension of S. Typhimurium strain IR715 or delta sipA sopABDE2 mutant (ZA21) in LB broth containing approximately 0.75 X 10e9 CFU/ml. Loops injected with sterile LB broth (3 ml) served as negative controls. The loops were replaced into the abdominal cavity until collected by excision. At each of seven time points (15 min, 30 min, 1, 2, 4, 8, and 12 hours) three loops (one LB inoculated control loop, one wild type inoculated loop and one mutant inoculated loop) were excised.
Growth protocol
Derivatives of Salmonella enterica serotype Typhimurium strain ATCC 14028, IR715 and ZA21, were used in this study. IR715 is a spontaneous naldixic acid resistant derivative and ZA21 is a previously described delta sipA sopABDE2 mutant. Cultures were grown in Luria-Bertani (LB) broth [supplemented with appropriate antibiotics: naldixic acid (50 mg/l), kanamycin (100 mg/l), chloramphenicol (30 mg/l), and tetracycline (20 mg/l)]. To prepare the bacterial inoculum, wild type and mutant were cultured in LB broth for 18 hours at 37°C with shaking at 150 rpm in a shaking incubator (Model C24, New Brunswick Scientific, Edison, NJ). The resultant cultures were diluted 1:100 in sterile LB broth and incubated as before for an additional 4 hours. Bacteria in the exponential phase of growth were then harvested by centrifugation for 15 min at 1,500 Xg and resuspended in fresh LB broth to obtain a final concentration of approximately 0.75 x 10e9 colony-forming units/ml (CFU)/ml. The bacterial concentration of the inoculum was confirmed by spreading serial 10-fold dilutions on agar plates containing the appropriate antibiotics.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted after dissection of the ileal loops obtained at 15 min and 30 min and at 1, 2, 4, 8, and 12 hours post-infection. A 6 mm biopsy punch was used to collect 8 tissue pieces of Peyer’s patch from the excised loop. The mucosa of the samples were immediately dissected, the tissue pieces finely minced with a scalpel blade, and two biopsy punches (0.1 mg of tissue) transferred to 1 ml of Trizol reagent (Molecular Research Center, Cincinnati, OH). Tissues were further disrupted with an RNase, DNase free plastic disposable pestle. The RNA extraction was performed using the recommended protocol from the manufacturer. The resultant RNA pellet was re-suspended in DEPC-treated water (Ambion, Austin, TX). Genomic DNA was removed by RNase-free DNase I treatment (DNA-free, Ambion) according to the manufacture’s instructions, and samples were stored at −80ºC until used. The quality of the RNA was initially assessed by measurement of the lambda 260/280 ratio and agarose gel electrophoresis. RNA concentration was quantified by measuring absorbance at lambda 260nm using a NanoDrop® ND-1000 (NanoDrop, Wilmington, DW), and the RNA quality was determined using a Nano-Chip® on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). The RNA analyzed in all samples was of good to excellent quality (results not shown). The average RNA intensity number (RIN) was 7.42 and the median was 7.7. Samples were characterized by distinct 18S and 28S rRNA peaks and RNA size distribution. Ten micrograms of RNA was used as starting material for each array.To generate the reference RNA (which was labeled with Cy3), total RNA was isolated from Madin-Darby bovine kidney (MDBK) and bovine B lymphocyte (BL-3) cell lines (American Type Culture Collection, Manassas, VA) and fresh bovine brain cortex and cerebellum collected from surgery calves following euthanasia at the conclusion of each experiment. Cell lines were grown in 150 cm2 cell culture flasks with Eagle’s minimum essential medium (E-MEM) (ATCC) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS). Tissues were homogenized in ice-cold Tri-reagent® (Ambion, Austin, TX). RNA concentration and quality from each sample was determined before and after pooling the samples. Total RNA isolated from three samples was pooled together in equal amounts, aliquoted and stored at −80ºC until needed.
Label
Cy5
Label protocol
Briefly, cDNA from bovine experimental samples (i.e. from infected and control loops) and cDNA generated from the bovine reference RNA sample were co-hybridized to the previously described custom 13K bovine 70-mer oligoarray. The cDNA was reverse-transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and labeled with amino-allyl-UTP (Ambion, Austin, TX). Cy3 and Cy5 dye esters were covalently linked to the amino-allyl group by incubating the samples with the dye esters in 0.1M sodium carbonate buffer.
A mixture of equal parts total RNA from Madin-Darby bovine kidney (MDBK) and bovine B lymphocyte (BL-3) cell lines and fresh bovine brain cortex and cerebellum. cell line: Madin-Darby bovine kidney (MDBK) cell line: bovine B lymphocyte (BL-3) c tissue: fresh bovine brain cortex and cerebellum cell lines were purchased from the American Type Culture Collection (ATCC), fresh bovine brain and cerebellum was collected from surgery calves immediately following euthanasia by barbituate overdose at the conclusion of bovine ligated ileal loop surgery.
Treatment protocol
The bovine ligated ileal loop surgical procedure has been described. Briefly, the calves were fasted for 12 hours prior to the surgery. Anesthesia was induced with propofol (Abbot Laboratories, Chicago, IL) followed by placement of an endotracheal tube and maintainance with isofluorane (Isoflo, Abbot Laboratories) for the duration of the experiment. A laparotomy was performed, the distal ileum was exposed, and 21 loops approximately 6 to 9 cm in length were ligated with umbilical tape, leaving 1 cm loops between them. Overnight bacterial cultures were grown as described above. The ileal loops were inoculated by intralumenal injection of 3 ml of a suspension of S. Typhimurium strain IR715 or delta sipA sopABDE2 mutant (ZA21) in LB broth containing approximately 0.75 X 10e9 CFU/ml. Loops injected with sterile LB broth (3 ml) served as negative controls. The loops were replaced into the abdominal cavity until collected by excision. At each of seven time points (15 min, 30 min, 1, 2, 4, 8, and 12 hours) three loops (one LB inoculated control loop, one wild type inoculated loop and one mutant inoculated loop) were excised.
Growth protocol
Derivatives of Salmonella enterica serotype Typhimurium strain ATCC 14028, IR715 and ZA21, were used in this study. IR715 is a spontaneous naldixic acid resistant derivative and ZA21 is a previously described delta sipA sopABDE2 mutant. Cultures were grown in Luria-Bertani (LB) broth [supplemented with appropriate antibiotics: naldixic acid (50 mg/l), kanamycin (100 mg/l), chloramphenicol (30 mg/l), and tetracycline (20 mg/l)]. To prepare the bacterial inoculum, wild type and mutant were cultured in LB broth for 18 hours at 37°C with shaking at 150 rpm in a shaking incubator (Model C24, New Brunswick Scientific, Edison, NJ). The resultant cultures were diluted 1:100 in sterile LB broth and incubated as before for an additional 4 hours. Bacteria in the exponential phase of growth were then harvested by centrifugation for 15 min at 1,500 Xg and resuspended in fresh LB broth to obtain a final concentration of approximately 0.75 x 10e9 colony-forming units/ml (CFU)/ml. The bacterial concentration of the inoculum was confirmed by spreading serial 10-fold dilutions on agar plates containing the appropriate antibiotics.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted after dissection of the ileal loops obtained at 15 min and 30 min and at 1, 2, 4, 8, and 12 hours post-infection. A 6 mm biopsy punch was used to collect 8 tissue pieces of Peyer’s patch from the excised loop. The mucosa of the samples were immediately dissected, the tissue pieces finely minced with a scalpel blade, and two biopsy punches (0.1 mg of tissue) transferred to 1 ml of Trizol reagent (Molecular Research Center, Cincinnati, OH). Tissues were further disrupted with an RNase, DNase free plastic disposable pestle. The RNA extraction was performed using the recommended protocol from the manufacturer. The resultant RNA pellet was re-suspended in DEPC-treated water (Ambion, Austin, TX). Genomic DNA was removed by RNase-free DNase I treatment (DNA-free, Ambion) according to the manufacture’s instructions, and samples were stored at −80ºC until used. The quality of the RNA was initially assessed by measurement of the lambda 260/280 ratio and agarose gel electrophoresis. RNA concentration was quantified by measuring absorbance at lambda 260nm using a NanoDrop® ND-1000 (NanoDrop, Wilmington, DW), and the RNA quality was determined using a Nano-Chip® on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). The RNA analyzed in all samples was of good to excellent quality (results not shown). The average RNA intensity number (RIN) was 7.42 and the median was 7.7. Samples were characterized by distinct 18S and 28S rRNA peaks and RNA size distribution. Ten micrograms of RNA was used as starting material for each array.To generate the reference RNA (which was labeled with Cy3), total RNA was isolated from Madin-Darby bovine kidney (MDBK) and bovine B lymphocyte (BL-3) cell lines (American Type Culture Collection, Manassas, VA) and fresh bovine brain cortex and cerebellum collected from surgery calves following euthanasia at the conclusion of each experiment. Cell lines were grown in 150 cm2 cell culture flasks with Eagle’s minimum essential medium (E-MEM) (ATCC) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS). Tissues were homogenized in ice-cold Tri-reagent® (Ambion, Austin, TX). RNA concentration and quality from each sample was determined before and after pooling the samples. Total RNA isolated from three samples was pooled together in equal amounts, aliquoted and stored at −80ºC until needed.
Label
Cy3
Label protocol
Briefly, cDNA from bovine experimental samples (i.e. from infected and control loops) and cDNA generated from the bovine reference RNA sample were co-hybridized to the previously described custom 13K bovine 70-mer oligoarray. The cDNA was reverse-transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and labeled with amino-allyl-UTP (Ambion, Austin, TX). Cy3 and Cy5 dye esters were covalently linked to the amino-allyl group by incubating the samples with the dye esters in 0.1M sodium carbonate buffer.
Hybridization protocol
Prior to hybridization, the microarrays were denatured by exposing to steam from boiling water for three seconds, UV cross-linked and then immersed in pre-hybridization buffer [5X sodium chloride, sodium citrate buffer (SSC), 0.1% sodium dodecyl sulfate (SDS) (Ambion, Austin, TX), 1% bovine serum albumin (BSA)] at 42ºC for a minimum of 45 min. The arrays were then washed four times in RNase-, DNase-free, distilled water, immersed in 100% isopropanol for 10 seconds, and dried by centrifugation. Slides were hybridized at 42ºC for approximately 40 hours in a dark, humid chamber (Corning, Corning, NY), washed for 10 min at 42ºC with low stringency buffer [1 X SSC, 0.2% SDS] and then washed twice for 5-min each wash in a higher stringency buffer [0.1 X SSC, 0.2% SDS and 0.1 X SSC] at room temperature with agitation.
Scan protocol
Immediately after washing, the slides were scanned using a commercial laser scanner (GenePix 4100; Axon Instruments Inc., Foster City, CA). Scans were performed using the autoscan feature with the percent saturated pixels set at 0.03%
Description
n/a
Data processing
The spots representing genes on the arrays were adjusted for background and normalized to internal controls using image analysis software (GenePixPro 4.0; Axon Instruments Inc.). Spots with fluorescent signal values below background were disregarded in all analyses. Samples were normalized against the bovine reference RNA signals across slides and within each slide. Genes were considered as significantly altered if the average fold-change was at least 1.5, the adjusted p-value was less than 0.05, the alteration was reproducible across replicates, and the magnitude of difference between conditions was greater than 50% of the fold-change obtained for any two replicate controls.
