|
| Status |
Public on Feb 11, 2016 |
| Title |
Pooled_Tissue_UK [10 - 4G] |
| Sample type |
genomic |
| |
|
| Source name |
Pooled sample of liver, kidney and lung
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: Pooled sample of liver, kidney and lung
|
| Treatment protocol |
N/A
|
| Growth protocol |
N/A
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Automated nucleic acid extraction was performed on the samples using a QIAcube (Qiagen) and appropriate kit (cador Pathogen Mini Kit (Qiagen)), following partial homogenisation of each tissue sample. The protocol Purification of Pathogen Nucleic Acids from Fluid Samples (Qiagen) was used. This protocol is intended for the purification of viral RNA and DNA and the DNA of easy-to-lyse bacteria from fluid samples or pretreated tissue samples.
|
| Label |
Biotin
|
| Label protocol |
Biotin labelling was performed using biotinylated primers to produce a product ready for hybridisation
|
| |
|
| Hybridization protocol |
The arrays were conditioned by washing with 150 µl water for 20 minutes at 30°C followed by incubation with 500 µl of pre-hybridisation buffer (5x SSC (Life Technologies), 0.1% SDS (Sigma), 4x Denhardt's solution (Life Technologies)) for 30 minutes at 50°C. 10µl of the labelled DNA was added to 90µl hybridisation buffer (5x SSC, 1% SDS, 4x Denhardt's solution). The sample was denatured for 3 minutes at 95°C. The sample was allowed to hybridise with the microarray for 30 minutes at 50°C at 550rpm. The array was then washed with 150 µl Wash Buffer 1 (1x SSC, 0.2% SDS) for 20 minutes at 60°C at 550 rpm. The array was then washed with 150 µl Wash Buffer 2 (0.1% SSC, 0.2% SDS) for 20 minutes at 60°C at 550 rpm. The array was then washed with 150 µl Wash Buffer 3 (0.1x SSC) for 20 minutes at 60°C at 550 rpm. This buffer was removed and a blocking solution (100µl) of 2% biotin-free milk (Marvel) in PBS containing 1% BSA (New England Biolabs) and 0.1% Tween20 was added and incubated for 60 minutes at 30°C at 300 rpm. The blocking solution was removed and replaced with a conjugation solution (Streptavidin Poly-Horse radish peroxidase (HRP), ThermoScientific) diluted 1:100 in the blocing solution and incubated at 15 minutes at 30°C at 300 rpm. Post-conjugation washes with Wash Buffers 1, 2 and 3 were performed again. Following removal of Wash Buffer 3, 100 µl of the Tetramethylbenzidine couple with hydrogen peroxide (TMB/H2O2) staining solution (TrueBlue, Insight BioTechnology LTD) was added and incubated for 10 minutes at 25°C.
|
| Scan protocol |
The ArrayStrip was inserted into the ArrayMate (Alere) and the array image recorded.
|
| Description |
Co-purified RNA and DNA
|
| Data processing |
The recorded image was analysed using Alere's integrated Iconoclust software and analysis script. Iconoclust processes the signals and automatically normalises the signal value after an algorithm processes the average intensity of the spot and the local background noise.
|
| |
|
| Submission date |
Feb 10, 2016 |
| Last update date |
Feb 11, 2016 |
| Contact name |
Tim Giles |
| E-mail(s) |
timothy.giles@nottingham.ac.uk
|
| Organization name |
University of Nottingham
|
| Street address |
College Road
|
| City |
Loughborough |
| ZIP/Postal code |
LE12 5RD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21445 |
| Series (1) |
| GSE77765 |
Detection of a Yersinia pestis homologue in rodent samples |
|
