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| Status |
Public on Sep 20, 2008 |
| Title |
PBMC_CRR-vs-BTB-infected-tuberculin-stimulated_T3_1 |
| Sample type |
RNA |
| |
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| Channel 1 |
| Source name |
Peripheral blood mononuclear cells (PBMC) sampled from 13 animals across a tuberculin stimulation time course
|
| Organism |
Bos taurus |
| Characteristics |
Source: PBMC from eight M. bovis-infected cattle and five non-infected cattle. PBMC were stimulated with purified protein derivative (PPD) of Mycobacterium bovis (tuberculin) and sampled across a time course of T0 (no stimulation) T3 (3 hours post-stimulation) and T12 (12 hours post-stimulation). A total of 39 RNA samples were combined to form the common RNA reference (CRR) pool
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| Biomaterial provider |
Animal Genomics Laboratory, University College Dublin, Ireland.
|
| Treatment protocol |
PBMC cultures from the eight animals were stimulated using 50 µg/ml bovine purified protein derivative (PPD-bovine) and unstimulated control cultures from the same animals were run in parallel.
|
| Growth protocol |
A Trypan exclusion test was used to estimate cell numbers: 10 µl of the cell solution was added to 90 µl Trypan (100 ml trypan blue solution 0.4% - T8154, Sigma-Aldrich Ireland Ltd: www.sigmaaldrich.com) in a 96-well microtitre plate and left for 5 mins. Viable cells were counted by pipetting 10 µl of solution under the coverslip of a haemocytometer, using a light microscope. Cells from each animal were seeded at no less than 1E+07 for each of the two treatments and cultured in RPMI 1640 culture medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamycin. PBMC were cultured in 75-ml culture flasks at 37 °C in a humidified atmosphere with 5% CO2. PBMC cultures from the eight animals were stimulated using 50 µg/ml bovine purified protein derivative (PPD-bovine) and unstimulated control cultures from the same animals were run in parallel. PBMC were harvested from all treatments at 0, 3 and 12 h post-stimulation and stored at -80°C in 1 ml of Trizol® reagent (Invitrogen Corp: www.invitrogen.com).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from PBMC using a combined TriReagent®, DNase treatment and Qiagen RNeasy® method (Qiagen Ltd: www.qiagen.com). Cells were removed from the -80°C freezer and thawed quickly on ice, 400 µl of chloroform was added and shaken for 10 s. After a 2 min room temperature incubation, tubes were centrifuged on maximum speed for one min to separate the phases. The upper aqueous phase was transferred to a new 1.5 ml tube and an equal volume of isopropanol was added. After shaking and incubating at room temperature for 20 mins, the samples were centrifuged on maximum speed at 4°C for 20 mins. The liquid was then removed and the RNA pellet washed with 1 ml 70% ethanol (stored at -20°C) by centrifugation at 4°C for 6 mins. The ethanol was removed by careful aspiration and the pellet was allowed to air dry for 10 mins after which it was resuspended in 100 µl of nuclease free water and incubated at 55°C to ensure complete resuspension. To remove any residual DNA, 5 µl DNase (1 U/µl) was added and incubated at 37°C for 15 mins. The extracted RNA was then ready for further purification using the Qiagen RNeasy® mini kit according to the manufacturer’s instructions and eluted twice, in 40 µl and 30 µl of nuclease free water, and stored at -80°C.
|
| Label |
Cy5
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| Label protocol |
Each labelling reaction contained a total of 8 µg RNA per sample and 10 µg RNA from the CRR. Total RNA was used as the template in reverse transcription reactions (BD Atlas Powerscript fluorescent labelling system, BD Biosciences: www.bdbeurope.com) using oligo(dT) 15-18 as primer and incorporating amino-modified dUTPs. To provide a control for cDNA synthesis and labelling efficiency, hybridisation and slide orientation, 650 pg of synthetic lambda Q gene RNA containing an engineered poly A tail was spiked into each cDNA synthesis reaction. Test and CRR samples were differentially labelled using N-hydroxysuccinimide-(NHS) activated fluorescent Cy3 or Cy5 dyes (Amersham Biosciences/GE Healthcare: www.amershambiosciences.com). The dye couples to the cDNA by reacting with the amino functional groups on the incorporated amino-tagged dUTPs. Labelled cDNAs were purified to remove unincorporated dyes using a QIAquick purification kit and concentrated using Microcon® centrifugal filter devices according to the manufacturer’s instructions (Millipore Corp: www.millipore.com).
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| |
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| Channel 2 |
| Source name |
PBMC-BTB-infected-animal-Tuberculin-stimulated-T3-1
|
| Organism |
Bos taurus |
| Characteristics |
PBMC were stimulated with purified protein derivative (PPD) of Mycobacterium bovis (tuberculin) and sampled 3 hours post-stimulation (time 'T3)
|
| Biomaterial provider |
Animal Genomics Laboratory, University College Dublin, Ireland.
|
| Treatment protocol |
PBMC cultures were stimulated using 50 µg/ml bovine purified protein derivative (PPD-bovine).
|
| Growth protocol |
PBMC from each animal (control and infected) were seeded at 1E+07 per culture plate and cultured in RPMI 1640 culture medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamycin. PBMC were cultured in 75-ml culture flasks at 37 °C in a humidified atmosphere with 5% CO2. Overnight culture was carried out to minimise noise in gene expression measurements potentially introduced by the mechanical disruption of cells associated with PBMC isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from PBMC using a combined TriReagent®, DNase treatment and Qiagen RNeasy® method (Qiagen Ltd: www.qiagen.com). Cells were removed from the -80°C freezer and thawed quickly on ice, 400 µl of chloroform was added and shaken for 10 s. After a 2 min room temperature incubation, tubes were centrifuged on maximum speed for one min to separate the phases. The upper aqueous phase was transferred to a new 1.5 ml tube and an equal volume of isopropanol was added. After shaking and incubating at room temperature for 20 mins, the samples were centrifuged on maximum speed at 4°C for 20 mins. The liquid was then removed and the RNA pellet washed with 1 ml 70% ethanol (stored at -20°C) by centrifugation at 4°C for 6 mins. The ethanol was removed by careful aspiration and the pellet was allowed to air dry for 10 mins after which it was resuspended in 100 µl of nuclease free water and incubated at 55°C to ensure complete resuspension. To remove any residual DNA, 5 µl DNase (1 U/µl) was added and incubated at 37°C for 15 mins. The extracted RNA was then ready for further purification using the Qiagen RNeasy® mini kit according to the manufacturer’s instructions and eluted twice, in 40 µl and 30 µl of nuclease free water, and stored at -80°C.
|
| Label |
Cy3
|
| Label protocol |
Each labelling reaction contained a total of 8 µg RNA per sample and 10 µg RNA from the CRR. Total RNA was used as the template in reverse transcription reactions (BD Atlas Powerscript fluorescent labelling system, BD Biosciences: www.bdbeurope.com) using oligo(dT) 15-18 as primer and incorporating amino-modified dUTPs. To provide a control for cDNA synthesis and labelling efficiency, hybridisation and slide orientation, 650 pg of synthetic lambda Q gene RNA containing an engineered poly A tail was spiked into each cDNA synthesis reaction. Test and CRR samples were differentially labelled using N-hydroxysuccinimide-(NHS) activated fluorescent Cy3 or Cy5 dyes (Amersham Biosciences/GE Healthcare: www.amershambiosciences.com). The dye couples to the cDNA by reacting with the amino functional groups on the incorporated amino-tagged dUTPs. Labelled cDNAs were purified to remove unincorporated dyes using a QIAquick purification kit and concentrated using Microcon® centrifugal filter devices according to the manufacturer’s instructions (Millipore Corp: www.millipore.com).
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| |
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| |
| Hybridization protocol |
Labelled samples were combined (either an infected or a control sample combined with a CRR sample) and co-hybridized on the BOTL-5 microarrays using SlideHyb Glass Array Hybridization Buffer #3 (Ambion Ltd.). Microarray hybridizations were performed using a Tecan HS400 hybridisation station (Tecan Ltd.) with the following protocol – Step 1: 75°C, wash 10 s, soak 20 s, 1 cycle; probe injection: 85°C; denaturation: 95°C, 2 min; hybridization cycle 1: 65°C, time 35 min, agitation frequency medium; hybridization cycle 2: 55°C, time 35 min, agitation frequency medium; hybridization cycle 3: 50°C, time 2 h 30m, agitation frequency medium; wash cycle 1: 42°C, wash 10 s, soak 20 s, 2 cycles; wash cycle 2: 33°C, wash 15 s, soak 30 s, 2 cycles; wash cycle 3: 33°C, wash 20 s, soak 40 s, 2 cycles; slide drying: 30°C, 1 min 30 s.
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| Scan protocol |
Microarrays were scanned immediately using a GenePix 4000B microarray scanner (Molecular Devices Ltd.). Data was captured using GenePix Pro version 5.0 software (Molecular Devices Ltd.).
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| Description |
No additional information.
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| Data processing |
The working signal intensities were generated using the mean foreground intensity values minus the median background intensity values as outputted from the GenePix Pro 5.0 results file. Two methods of data pre-processing were used to flag unreliable data. If the signal intensity in one channel was less than 100 and if the signal intensity for the other channel is less than 200, the spot was flagged. If the signal intensity in one channel was less than 100 and the signal intensity was larger than 200 in the second channel, 100 was assigned to the intensity of the first channel. Median-based normalization corrects the data such that all arrays have the same median. The median value is less likely to be influenced by outlying values. A normalization factor was calculated by summing the intensities in both channels and adjusts both ratios to ensure a median of 1.0. Therefore, the median log expression ratio for all features on the array was adjusted to zero (corresponding to an expression ratio of 1.0).
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| Submission date |
Sep 17, 2008 |
| Last update date |
Sep 19, 2008 |
| 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 |
| |
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| Platform ID |
GPL5751 |
| Series (1) |
| GSE12835 |
Comparison of antigen stimulation response in Mycobacterium bovis-infected vs. control cattle using BOTL-5 microarray |
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