tissue: monocyte-derived macrophage cattle breed: Holstein-Friesian Sex: Female age: 3 years infection: none time point: 2 hr
Treatment protocol
All challenge experiments including the preparation of non-challenge control samples were performed in the CL3 laboratory. Prior to the M. bovis challenge experiments, MDMs from two adjacent culture plate wells were lysed using RLT buffer from the RNeasy Mini kit (Qiagen Ltd., Crawley, UK) supplemented with 1% Beta-mercaptoethanol (Sigma-Aldrich Ireland Ltd., Dublin, Ireland) and pooled. These samples constituted the 0 hour control MDM samples. MDM samples (seeded at 2E+5 cells per well) were challenged with M. bovis (4E+5 cells/well; based on bacterial cell counts performed using a Petroff Hausser chamber (Fisher-Scientific, Dublin, Ireland) [multiplicity of infection 2:1] and incubated at 37°C, 5% CO2 for 2 hours, 6 hours and 24 hours. For the non-challenged control samples at each time-point, antibiotic-free culture media (RPMI 1640 medium containing 15% heat inactivated FCS and 1% NEAA only) was added to each well. After 2 hours post-challenge, the media from all 6 hour and 24 hour challenge experiments was replaced with 0.5 ml fresh antibiotic-free culture media per well and re-incubated at 37°C, 5% CO2 until MDM were required for harvesting. Challenged and non-challenged control MDMs were lysed and harvested using RLT/1% Beta-mercaptoethanol buffer (Qiagen Ltd., Crawley, UK) at the designated time points. For each treatment, MDM lysates from two culture plate wells were pooled and stored at 80°C until required for RNA extraction.
Growth protocol
For monocyte isolation, 300 ml of whole blood was collected in acid citrate dextrose buffer (Sigma-Aldrich Ireland Ltd., Dublin, Ireland) in sterile bottles. Blood was layered onto Accuspin™ tubes containing Histopaque® 1077 (Sigma-Aldrich Ireland Ltd., Dublin, Ireland) and following density gradient centrifugation, peripheral blood mononuclear cells (PBMC) were collected. Contaminating red blood cells (RBC) were removed following resuspension and subsequent incubation of the PBMC in RBC lysis buffer (10mM KHCO3, 150mM NH4Cl, 0.1mM EDTA pH 8.0) for 5 minutes at room temperature. After incubation, PBMC were washed twice with phosphate-buffered saline (PBS) before resuspending cells in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA, Sigma-Aldrich Ireland Ltd., Dublin, Ireland). Monocytes were then isolated using the MACS protocol and a human anti-CD14 antibody (Miltenyi Biotec Ltd., Surrey, UK), which has been shown to be cross-reactive with bovine monocytes. All procedures were carried out according to the manufacturers’ instructions. The identity and purity of monocytes was confirmed by flow cytometry using an anti-CD14 FITC labelled antibody (data not shown). This method has been previously shown by us to yield a purity of CD14+ cells >= 99%. Purified monocytes were seeded at 1+E6/ml in 24-well tissue culture plates in RPMI 1640 medium (Invitrogen Ltd., Paisley, UK) containing 15% heat inactivated foetal calf serum (FCS; Sigma-Aldrich Ireland Ltd., Dublin, Ireland), 1% non-essential amino acids (NEAA; Sigma-Aldrich Ireland Ltd., Dublin, Ireland), gentamicin (5 µg/ml; Sigma-Aldrich Ireland Ltd., Dublin, Ireland) and incubated at 37°C, 5% CO2. Following 24 hours incubation (day one) the media was replaced with 1ml fresh antibiotic-containing media to remove any non-adhered cells. On day three, media was replaced with 1 ml antibiotic-free culture media (RPMI 1640 medium containing 15% heat inactivated FCS and 1% NEAA only). To ensure that the same number of MDM were subjected to different treatments between experiments, cells were dissociated on day five using a non-enzymatic cell dissociation solution (Sigma-Aldrich Ireland Ltd., Dublin, Ireland), counted and then re-seeded at 2+E5 cells/well in 24-well tissue culture plates (Sarstedt, County Wexford, Ireland) using antibiotic-free culture media. By day eight, 80-100% confluent monolayers of MDM were generated that displayed the characteristic macrophage morphology as confirmed by Giemsa staining. Day eight MDM were used for the in vitro challenge experiments with M. bovis.
Extracted molecule
total RNA
Extraction protocol
All RNA extractions were performed in the CL3 laboratory using an RNeasy kit incorporating an on-column DNase treatment step (Qiagen Ltd., Crawley, UK) according to the manufacturer’s instructions. RNA quantity and quality was assessed using both the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies, Cork, Ireland). All samples displayed a 260/280 ratio greater than 2.0 and RNA integrity numbers (RIN) greater than 8.5. cDNA labelling, hybridisation and scanning for the microarray experiments were performed using a one-cycle amplification/labelling protocol on the Affymetrix GeneChip Bovine Genome Array (http://www.affymetrix.com). This array represents over 23,000 transcripts and includes approximately 19,000 UniGene clusters.
Label
biotin
Label protocol
Total RNA was amplified using the NuGEN™ Ovation™ RNA Amplification System V2. First-strand synthesis of cDNA was performed using a unique first-strand DNA/RNA chimeric primer mix, resulting in cDNA/mRNA hybrid molecules. Following fragmentation of the mRNA component of the cDNA/mRNA molecules, second-strand synthesis was performed and double-stranded cDNA was formed with a unique DNA/RNA heteroduplex at one end. In the final amplification step, RNA within the heteroduplex was degraded using RNaseH, and replication of the resultant single-stranded cDNA was achieved through DNA/RNA chimeric primer binding and DNA polymerase enzymatic activity. The amplified single-stranded cDNA was purified for accurate quantitation of the cDNA and to ensure optimal performance during the fragmentation and labelling process. The single stranded cDNA was assessed using spectrophotometric methods in combination with the Agilent Bioanalyzer. The appropriate amount of amplified single-stranded cDNA was fragmented and labelled using the FL-Ovation™ cDNA Biotin Module V2. The enzymatically and chemically fragmented product (50-100 nt) was labelled via the attachment of biotinylated nucleotides onto the 3'-end of the fragmented cDNA.
Hybridization protocol
Fragmented and labelled cDNA was added to the hybridisation cocktail in accordance with the NuGEN™ guidelines for hybridisation onto Affymetrix GeneChip® arrays. Following the hybridisation for 16-18 hours at 45°C in an Affymetrix GeneChip® Hybridisation Oven 640, the array was washed and stained on the GeneChip® Fluidics Station 450 using the appropriate fluidics script, before being inserted into the Affymetrix autoloader carousel and scanned using the GeneChip® Scanner 3000.
Scan protocol
All Affymetrix Bovine Genome Array microarrays were scanned using an Affymetrix GeneChip® Scanner 3000.
Description
gene expression data from bovine monocyte-derived macrophage non-challenged at T2 rep6
Data processing
Affymetrix® GeneChip® Bovine Genome Array data were analysed using Bioconductor [http://www.bioconductor.org] contained within the R statistical package (http://www.r-project.org). Normalisation of raw data was performed using the Factor Analysis for Robust Microarray Summarization (FARMS) algorithm. In total, 89 arrays were used for normalisation, including 20 arrays from Mycobacterium avium subspecies paratuberculosis (MAP) stimulated MDM across the same time course and 20 arrays from M. bovis (BCG) stimulated MDM across the same time course. The FARMS algorithm uses only perfect match probes and a quantile normalization procedure, which provides the signal intensities.
Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis
