Supernatant from the monocyte-derived macrophages cultures was taken out and cells rinsed once with cold PBS at 4 and 12 h p.i. One ml of Tri-reagent® (Ambion, Austin, TX) was added to each flask and RNA extracted using the recommended protocol from the manufacturer. The resultant RNA pellet was re-suspended in DEPC-treated water (Ambion) with 1% RNAse inhibitor (Promega, Madison, WI). Genomic DNA was removed by RNase free - DNAse I treatment (Ambion) according to the manufacturer’s instructions, and samples were stored at -80ºC until used.
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 a 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.
tissue: brain cortex and cerebellum cell line: Madin-Darby bovine kidney (MDBK) and bovine B lymphocyte (BL-3)
Biomaterial provider
Cell lines were purchased from the American Type Culture Collection (ATCC), fresh bovine brain and cerebellum were collected from surgery calves immediately following euthanasia by barbituate overdose at the conclusion of bovine ligated ileal loop surgery.
Extracted molecule
total RNA
Extraction protocol
Supernatant from the monocyte-derived macrophages cultures was taken out and cells rinsed once with cold PBS at 4 and 12 h p.i. One ml of Tri-reagent® (Ambion, Austin, TX) was added to each flask and RNA extracted using the recommended protocol from the manufacturer. The resultant RNA pellet was re-suspended in DEPC-treated water (Ambion) with 1% RNAse inhibitor (Promega, Madison, WI). Genomic DNA was removed by RNase free - DNAse I treatment (Ambion) according to the manufacturer’s instructions, and samples were stored at -80ºC until used.
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 a 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
Reference RNA is 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.
S 4h D control
Data processing
The spots representing genes on the arrays were adjusted for background and normalized to internal controls using image analysis software (GenePix Pro 6.0; Axon Instruments, Inc.). Spots with fluorescent signal values below background were disregarded in all analyses. Initially, arrays were normalized against bovine reference RNA. Data was analyzed using GeneSifter (VizX Labs, Seattle, WA) and a combinatorial statistical approach as follows: data were normalized by mean and then combined, Student’s t test was performed with a cutoff value of 0.05, and pairwise comparisons were made with a fold-change cutoff of 1.5 or 2-fold. Computational hierarchical cluster analysis, analysis of variance (ANOVA), and principal component analysis (PCA) were performed using Spotfire DecisionSite 8.2 (Spotfire, Inc., Somerville, MA), after normalization by mean.
