|
| Status |
Public on Dec 01, 2011 |
| Title |
Peripheral blood leukocytes taken from Mycobacterium bovis infected cattle. Rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood leukocytes taken from Mycobacterium bovis infected cattle
|
| Organism |
Bos taurus |
| Characteristics |
tissue: Peripheral blood leukocyte
cattle breed: Holstein-Friesian
Sex: Female
infection status: Infected
agent: Mycobacterium bovis
|
| Treatment protocol |
Sixteen animals were used for this study. Eight infected animals were selected from a panel of naturally M. bovis infected animals maintained for on-going disease surveillance at the Department of Agriculture, Fisheries and Food, Backweston Laboratory Campus (Celbridge, Co. Kildare, Ireland). These animals had a positive single intradermal comparative tuberculin test (SICTT) result and were also positive in the whole blood IFN-γ-based BoviGAM® assay [Prionics AG, Switzerland] (data not shown). Cattle were confirmed for BTB following detailed post-mortem pathological examination. Non-infected controls animals were sex matched and age-matched to the infected animals and were selected from a herd with no recent history of M. bovis infection. The control animals were shown to be negative for both the SICTT and IFN-γ tests (data not shown).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All RNA extractions were performed within two hours of blood collection. Briefly, 7.5 ml of whole heparinised blood was mixed with 42.5 ml of erythrocyte blood lysis buffer (10 mM KHCO3, 150 mM NH4Cl, 1 mM EDTA pH 8.0), and incubated for 5 min at room temperature with gentle agitation. Following centrifugation (750g for 10 min) the pelleted cells were washed once with 1× phosphate buffered saline (Invitrogen Ltd., Paisley, UK). The cell pellet was then fully resuspended in 2 ml Trizol® reagent (Invitrogen Ltd., Paisley, UK) and RNA was extracted as per the manufacturer’s instructions. The RNA was further purified using an RNeasy® kit with on-column DNase treatment (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 Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies, Cork, Ireland). All samples displayed a 260/280 ratio greater than 1.8 and RNA integrity numbers (RIN) greater than 8.0.
|
| 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.
|
| 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. The FARMS algorithm uses only perfect match probes and a quantile normalization procedure, which provides the signal intensities.
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| |
|
| Submission date |
Nov 01, 2011 |
| Last update date |
Dec 01, 2011 |
| Contact name |
David E MacHugh |
| E-mail(s) |
david.machugh@ucd.ie
|
| Phone |
+353-1-7166256
|
| Organization name |
University College Dublin
|
| Department |
College of Agriculture, Food Science and Veterinary Medicine
|
| Lab |
Animal Genomics Laboratory
|
| Street address |
University College Dublin, Belfield
|
| City |
Dublin |
| ZIP/Postal code |
D4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL2112 |
| Series (1) |
| GSE33359 |
Genome-wide transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis |
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