|
| Status |
Public on Mar 31, 2021 |
| Title |
Brahman R microplus larval feeding replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Brahman superficial cervical lymph node, R microplus larval feeding
|
| Organism |
Bos taurus |
| Characteristics |
tissue: Lymph node
breed: Brahman
sample type: Rhipicephalus microplus larvae tick attachment
|
| Treatment protocol |
Lymph node biopsies were collected, kept on ice and transferred to an RNA stabilizing solution (0.5 M EDTA, 1 M ammonium citrate, 5.3 M ammonium sulphate, adjusted to pH 5.2 with H2SO4).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRI-reagent fractionation (Sigma-Aldrich) and the RNeasy mini kit (QIAGEN, Germany) following the manufacturer’s instructions. Contaminating genomic DNA was removed with a DNase I treatment (QIAGEN, Germany).
|
| Label |
Cy5
|
| Label protocol |
First-strand cDNA synthesis was performed by incubating 4 µg RNA (from each biological replicate and the reference pool) with 250 pmol oligo(dT25) and 775 pmol random primer 9 for 10 minutes at 70˚C, followed by cooling on ice for 10 minutes. Reverse transcription and aminoallyl-dUTP (5-(3-aminoallyl)-2’deoxyuridine-5’ triphosphate) incorporation were performed simultaneously using 340 units SuperScript® III reverse transcriptase (Invitrogen™ life technologies, USA).
cDNA samples were coupled to 75 picomol Cy3 (reference pool) and Cy5 (samples) fluorescent dyes (GE Healthcare Life Sciences)at pH 9 dissolved in DMSO. Unincorporated dye was then removed.
|
| |
|
| Channel 2 |
| Source name |
Pooled cattle superficial cervical lymph nodes
|
| Organism |
Bos taurus |
| Characteristics |
tissue: Lymph node
|
| Treatment protocol |
Lymph node biopsies were collected, kept on ice and transferred to an RNA stabilizing solution (0.5 M EDTA, 1 M ammonium citrate, 5.3 M ammonium sulphate, adjusted to pH 5.2 with H2SO4).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRI-reagent fractionation (Sigma-Aldrich) and the RNeasy mini kit (QIAGEN, Germany) following the manufacturer’s instructions. Contaminating genomic DNA was removed with a DNase I treatment (QIAGEN, Germany).
|
| Label |
Cy3
|
| Label protocol |
First-strand cDNA synthesis was performed by incubating 4 µg RNA (from each biological replicate and the reference pool) with 250 pmol oligo(dT25) and 775 pmol random primer 9 for 10 minutes at 70˚C, followed by cooling on ice for 10 minutes. Reverse transcription and aminoallyl-dUTP (5-(3-aminoallyl)-2’deoxyuridine-5’ triphosphate) incorporation were performed simultaneously using 340 units SuperScript® III reverse transcriptase (Invitrogen™ life technologies, USA).
cDNA samples were coupled to 75 picomol Cy3 (reference pool) and Cy5 (samples) fluorescent dyes (GE Healthcare Life Sciences)at pH 9 dissolved in DMSO. Unincorporated dye was then removed.
|
| |
|
| |
| Hybridization protocol |
Equivalent picomoles (20 pmol) of Cy3-labelled cDNA from the common reference pool were hybridised with Cy5-labelled individual test cDNA. Overnight hybridisation at 65°C (rotation speed of 10), washing and post-processing were performed at the ACGT Microarray Facility (University of Pretoria, South Africa). Prior to slide scanning with the Axon GenePix 4000B scanner (Molecular Devices), slides were washed and dried by centrifugation
|
| Scan protocol |
Scanned on an Axon GenePix 4000B scanner (Molecular Devices)
Images were measured, recorded, analysed and spots were inspected using Axon GenePix Pro 6.0 software (Molecular Devices)
|
| Description |
Biological replicate 2 of 3. Brahman lymph node tissue. Rhipicephalus microplus larvae tick attachment.
|
| Data processing |
Axon GenePix Pro 6.0 software (Molecular Devices) and the linear model for microarray data analysis (LIMMA) within the R statistical environment (http://cran.r-project.org/) was employed. Adaptive background correction (offset = 50) was followed by within-array normalisation (Robust Spline) and between-array normalisation (G quantile). Fold change was determined between all transcripts within a cattle breed collected at different time-points using the empirical Bayesian statistics, which were subsequently expressed as P- values (corrected for false discovery rate).
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| |
|
| Submission date |
Apr 01, 2020 |
| Last update date |
Mar 31, 2021 |
| Contact name |
Dave Kenneth Berger |
| E-mail(s) |
[email protected]
|
| Phone |
+27124204634
|
| Organization name |
University of Pretoria
|
| Department |
Plant and Soil Science
|
| Lab |
Molecular and Plant Pathogen Interactions
|
| Street address |
Lynnwood Road
|
| City |
Pretoria |
| State/province |
Gauteng |
| ZIP/Postal code |
0002 |
| Country |
South Africa |
| |
|
| Platform ID |
GPL11648 |
| Series (1) |
| GSE147918 |
Cattle lymph nodes: Control vs. Larva tick-infested; Control vs. Adult tick-infested; Larva tick-infested vs. Adult tick-infested |
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