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Sample GSM764008 Query DataSets for GSM764008
Status Public on Jul 20, 2011
Title Bacterial nucleic acid sample BMA_HPA_002
Sample type other
 
Source name In vitro grown bacterial cell culture
Organism Haemophilus influenzae
Characteristics strain: Hi2
extracted molecule: total nucleic acids
cell/tissue type: In vitro grown bacterial cell culture
Treatment protocol N/A
Growth protocol Bacterial cells cultured overnight on 5% horse blood/Brain Heart Infusion agar plates at 37 oC
Extracted molecule other
Extraction protocol Bacterial colonies were isolated and re-suspended in 200 ul TEG buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 2% glucose). 500 ul Guanidine hydrochloride lysis buffer (8 M guanidine hydrochloride, 0.2% sodium sarkinosate) was added to the bacterial suspension which was then mixed by inversion, 200 ul of chloroform was added to the emulsion and centrifuged at 13000 g for 10 minutes. The DNA was recovered from the upper aqueous layer by precipitation with 100 % ethanol and collected by centrifugation. After washing twice with 70 % ethanol and drying, the DNA pellet was re-suspended in TE buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA) and stored at -20 oC prior to use.
Label Cy3
Label protocol The method used to generate randomly amplified DNA targets is a modification of the method published by Bohlander et al and has been described previously (see references 1 and 2 below)
Reference 1: Bohlander SK, Espinosa R 3rd, Le Beau MM, Rowley JD, Diaz MO. 1992 A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics. 13:1322-4.
Reference 2: Burton, J. E., O. J. Oshota, E. North, M. J. Hudson, N. Polyanskaya, J. Brehm, G. Lloyd, and N. J. Silman. 2005. Development of a multi-pathogen oligonucleotide microarray for detection of Bacillus anthracis. Mol Cell Probes 19:349-57
 
Hybridization protocol Slides were pre-hybridised in 5 x SSC, 0.1% SDS and 4 x Denhardts solution, washed in sterile, nuclease-free water followed by 100% propan-2-ol then air-dried. Cy3-labelled randomly-amplified target DNAs were denatured at 95 oC for 3 minutes then diluted to a final concentration of 80 μg/ml in 5 x SSC buffer (Sigma-Aldrich), 0.1% SDS, 4 x Denhardts solution at 50oC. 40 μl of hybridisation mix was applied to the microarray slide, which was then incubated in a humidified multi-slide chamber (Genetix Inc.) for 16 hours. The slides were then washed once in each of the following buffers for two minutes: (A) 1 x SSC, 0.2% SDS, 50oC, (B) 0.1 x SSC, 0.2% SDS, 50oC, (C) 0.1 x SSC, 0.2% SDS, 20oC, then centrifuged to dryness at 1000 rpm for 5 minutes
Scan protocol scanned using an Affymetrix 428 microarray laser scanner at a gain of sixty
Data processing After image capture files were saved in Tiff format then quantified using the scan analysis software Bluefuse™ (BlueGnome Inc. Cambridge UK), all data were normalised to the global median for each slide then replicate data points fused according to median fluorescent intensity and name. The data were sorted into pathogen relevant groups and each group analysed for statistical significance. Probe intensities were derived for each sample, the log(2) taken and p values calculated which were then sorted from lowest to highest values within each group. Data points for each group were then ranked first by decreasing fold intensity over human DNA control hybridisation values and then by significance (only oligonucleotides with p < 0.2 in each group were considered).
 
Submission date Jul 19, 2011
Last update date Jul 20, 2011
Contact name Karen Elizabeth Kempsell
E-mail(s) karen.kempsell@ukhsa.gov.uk
Phone ++44(0)1980 619816
Organization name UK Health Security Agency
Department Science Group
Lab Diagnostic Technologies
Street address Porton Down
City Salisbury
State/province Wiltshire
ZIP/Postal code SP4 0JG
Country United Kingdom
 
Platform ID GPL13948
Series (1)
GSE30794 Design of a Diagnostic Pan-Pathogen Oligonucleotide Microarray including a sub-Array for Detection of Meningitis-Causing Bacterial Pathogens

Data table header descriptions
ID_REF
VALUE Bluefuse™ analysis software-computed normalised to global median using Cy3 signal intensity, replicate data points fused on median and name.

Data table
ID_REF VALUE
1145789-1-A01Foot-and-mouthdiseasevirusO 75.701
1145789-1-A02Foot-and-mouthdiseasevirusO 71.348
1145789-1-A03Alcelaphineherpesvirus1 43.118
1145789-1-A04Alcelaphineherpesvirus1 80.684
1145789-1-A05Acutebeeparalysisvirus 57.233
1145789-1-A06Acutebeeparalysisvirus 152.883
1145789-1-A07Gallidherpesvirus3 20.336
1145789-1-A08Humanparainfluenzavirus3 40.191
1145789-1-A09Humanparainfluenzavirus3 22.846
1145789-1-A10Echovirus5 37.172
1145789-1-A11Lelystadvirus 14.319
1145789-1-A12NewcastlediseasevirusB1 2331.982
1145789-1-B01Foot-and-mouthdiseasevirusO 97.116
1145789-1-B02Foot-and-mouthdiseasevirusO 30.627
1145789-1-B03Alcelaphineherpesvirus1 38.541
1145789-1-B04Lactatedehydrogenase-elevatingvirus 40.759
1145789-1-B05Acutebeeparalysisvirus 136.171
1145789-1-B06Gallidherpesvirus3 129.71
1145789-1-B07Gallidherpesvirus3 138.145
1145789-1-B08Humanparainfluenzavirus3 49.522

Total number of rows: 2177

Table truncated, full table size 68 Kbytes.




Supplementary file Size Download File type/resource
GSM764008_H._influenzae_Hi2.txt.gz 638.4 Kb (ftp)(http) TXT
GSM764008_H._influenzae_Hi2_Normalised.txt.gz 132.7 Kb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file

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