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Sample GSM764036 Query DataSets for GSM764036
Status Public on Jul 20, 2011
Title Bacterial nucleic acid sample BMA_HPA_030
Sample type other
Source name In vitro grown bacterial cell culture
Organism Neisseria lactamica
Characteristics strain: 2006
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
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
VALUE Bluefuse™ analysis software-computed normalised to global median using Cy3 signal intensity, replicate data points fused on median and name.

Data table
1145789-1-A01Foot-and-mouthdiseasevirusO 1045.032
1145789-1-A02Foot-and-mouthdiseasevirusO 262.405
1145789-1-A03Alcelaphineherpesvirus1 543.121
1145789-1-A04Alcelaphineherpesvirus1 938.078
1145789-1-A05Acutebeeparalysisvirus 536.542
1145789-1-A06Acutebeeparalysisvirus 519.141
1145789-1-A07Gallidherpesvirus3 436.754
1145789-1-A08Humanparainfluenzavirus3 263.572
1145789-1-A09Humanparainfluenzavirus3 535.105
1145789-1-A10Echovirus5 1636.188
1145789-1-A11Lelystadvirus 346.209
1145789-1-A12NewcastlediseasevirusB1 257.419
1145789-1-B01Foot-and-mouthdiseasevirusO 338.231
1145789-1-B02Foot-and-mouthdiseasevirusO 257.06
1145789-1-B03Alcelaphineherpesvirus1 296.995
1145789-1-B04Lactatedehydrogenase-elevatingvirus 283.65
1145789-1-B05Acutebeeparalysisvirus 1164.178
1145789-1-B06Gallidherpesvirus3 9388.842
1145789-1-B07Gallidherpesvirus3 1437.022
1145789-1-B08Humanparainfluenzavirus3 1058.187

Total number of rows: 2177

Table truncated, full table size 70 Kbytes.

Supplementary file Size Download File type/resource
GSM764036_N._lactamica_2006.txt.gz 660.2 Kb (ftp)(http) TXT
GSM764036_N._lactamica_2006_Normalised.txt.gz 137.2 Kb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file

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