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Sample GSM2058839 Query DataSets for GSM2058839
Status Public on Feb 11, 2016
Title Pooled_Tissue_UK [9 - 12E]
Sample type genomic
 
Source name Pooled sample of liver, kidney and lung
Organism Apodemus sylvaticus
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

Data table header descriptions
ID_REF
VALUE Normalised signal values were obtained after the Iconoclust software ran the analysis script.

Data table
ID_REF VALUE
1 0.002467
2 -0.006828
3 0.008036
4 -0.000069
5 0.000727
6 -0.007198
7 0.000221
8 0.001751
9 0.007976
10 0.001864
11 0.003046
12 -0.002725
13 -0.001567
14 -0.00256
15 0.004849
16 0.00501
17 -0.006462
18 0.004524
19 0.003929
20 0.005746

Total number of rows: 654

Table truncated, full table size 8 Kbytes.




Supplementary file Size Download File type/resource
GSM2058839_9_-_12E.txt.gz 11.6 Kb (ftp)(http) TXT
Processed data included within Sample table

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