male calf 3 to 6 weeks of age outbred distal ileum
Treatment protocol
Cows were infected (ligated ileal loops) with PBS, a virulent strain of Mycobacterium avium (subsp. paratuberculosis) or an avirulent M. avium strain (subsp. avium) for 30 min, 1h, 2h, 4h, 8h, or 12h. The experiment was performed in quadruplicate (except for the avirulent infections at 2 and 8 hours, which were performed three times each). Post-infection, RNA was collected, processed, and then hybridized to custom bovine gene expression arrays, each of which represented 13,258 transcripts spotted in duplicate. There were therefore 70 arrays in total.
Growth protocol
Mycobacterium avium subsp. paratuberculosis (ATCC 19698 from American Type Culture Collection, Manassas, VA), was grown aerobically in 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 2.5% (vol/vol) glycerol (Sigma Chemical Co., St. Louis, MO), oleic acid-albumin-dextrose-catalase (Difco Laboratories, Detroit, MI), 0.05% Tween 80 (Sigma Chemical Co., MO), and 2 mg of Mycobactin J (Allied Monitor, Inc., Fayette, MO.). Mycobacterium avium subsp. avium (ATCC 35716 from American Type Culture Collection, Manassas, VA) was grown in 7H9 broth supplemented with 2.5% (vol/vol) glycerol, oleic acid-albumin-dextrose-catalase and 0.05% Tween 80.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted after dissection of the ileal loops obtained at 30 min and at 1, 2, 4, 8, and 12 hours post-infection. A 6 mm biopsy punch was used to collect 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). 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. Reference RNA was a pool of Madin-Darby bovine kidney and bovine B lymphocyte cell lines and fresh bovine brain cortex and cerebellum. 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
Total RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and labeled with amino-allyl-UTP (Ambion, Austin, TX). Cy3 (Reference) and Cy5 (Experimental, i.e. PBS, MAp or MAA inoculated loop) 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 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
Cows were infected (ligated ileal loops) with PBS, a virulent strain of Mycobacterium avium (subsp. paratuberculosis) or an avirulent M. avium strain (subsp. avium) for 30 min, 1h, 2h, 4h, 8h, or 12h. The experiment was performed in quadruplicate (except for the avirulent infections at 2 and 8 hours, which were performed three times each). Post-infection, RNA was collected, processed, and then hybridized to custom bovine gene expression arrays, each of which represented 13,258 transcripts spotted in duplicate. There were therefore 70 arrays in total.
Growth protocol
Mycobacterium avium subsp. paratuberculosis (ATCC 19698 from American Type Culture Collection, Manassas, VA), was grown aerobically in 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 2.5% (vol/vol) glycerol (Sigma Chemical Co., St. Louis, MO), oleic acid-albumin-dextrose-catalase (Difco Laboratories, Detroit, MI), 0.05% Tween 80 (Sigma Chemical Co., MO), and 2 mg of Mycobactin J (Allied Monitor, Inc., Fayette, MO.). Mycobacterium avium subsp. avium (ATCC 35716 from American Type Culture Collection, Manassas, VA) was grown in 7H9 broth supplemented with 2.5% (vol/vol) glycerol, oleic acid-albumin-dextrose-catalase and 0.05% Tween 80.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted after dissection of the ileal loops obtained at 30 min and at 1, 2, 4, 8, and 12 hours post-infection. A 6 mm biopsy punch was used to collect 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). 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. Reference RNA was a pool of Madin-Darby bovine kidney and bovine B lymphocyte cell lines and fresh bovine brain cortex and cerebellum. 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
Total RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and labeled with amino-allyl-UTP (Ambion, Austin, TX). Cy3 (Reference) and Cy5 (Experimental, i.e. PBS, MAp or MAA inoculated loop) 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
cDNA from bovine experimental samples (i.e. from MAP and MAA infected and PBS control loops) were co-hybridized against cDNA generated from the bovine reference RNA sample 13K bovine 70mer oligoarray. 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 followed by four washes in RNase-, DNase-free, distilled water, immersion 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) and washed for 10 min at 42ºC with low stringency buffer [1 X SSC, 0.2% SDS] followed by two 5-min washes in a higher stringency buffer [0.1 X SSC, 0.2% SDS and 0.1 X SSC] at room temperature in dark with mild 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
Arrays were normalized by scaling against the average reference intensity value (i.e., average across all 70 arrays), normalized by global mean, and then log transformed before statistical analyses were performed. Signals flagged as “bad” by GenePix were removed across all arrays in order to ensure that subsequent analyses for each time point were comparable. Pairwise comparisons of averaged signal values and Student’s t test were performed using GeneSifter software (VizX Labs, Seattle, WA). A fold-change of at least 1.5-fold and a p value of 0.05 or less was expected for a difference in signal to be considered statistically significant. All possible individual pairwise comparisons between controls and infections (38 comparisons each for 30 min, 1h, 4h, and 12h post-infection, and 34 comparisons each for 2h and 87hr post-infection) were also performed using Spotfire DecisionSite software (Spotfire, Inc., Somerville, MA). Genes were further filtered using these various comparisons in order to ensure biological relevance (i.e., that observed differences were not the result of random variation between uninfected animals) and consistency (i.e., reproducibility across experiments). Because a relatively high level of variation between PBS-infected animals was observed (R2 values ranged from 6.2 to 9.1), the following criteria were used: 1. Biological relevance was determined as alterations between baseline and experiment (e.g., PBS-infected versus MAP-infected animals) that were at least 50% greater (on average) than the fold-change observed between any two baseline samples (i.e., PBS-infected versus PBS infected). 2. Consistency was defined as an average fold-change of at least 1.5 between each control (separately) and the four (or three) experimental replicates.
Bovine Peyer's Patch Infected with Mycobacterium avium ssp. paratuberculosis and Mycobacterium avium ssp. avium
Data table header descriptions
ID_REF
VALUE
Arrays were normalized by scaling against the average reference intensity value (i.e., average across all 70 arrays), normalized by global mean, and then log transformed before statistical analyses were performed. Signals flagged as bad by GenePix were removed across all arrays in order to ensure that subsequent analyses for each time point were comparable (normalized test value scaled against the average reference intensity value)
