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Sample GSM1955210 Query DataSets for GSM1955210
Status Public on Oct 08, 2016
Title challenge, 70 days [CH-70-50]
Sample type RNA
 
Channel 1
Source name lung
Organism Mus musculus
Characteristics treatment: challenge (secondary infection)
time: 70 days
Treatment protocol Three healthy mice were euthanized at day 0 and considered as naïve group. Mice assigned to the challenge groups were were intranasally infected under anesthesia (isoflurane/oxygen), with 2x10^5 cfu Bordetella pertussis B1917 in 40 µl Verweij medium at day 0. For both primary and secondary infections, mice were were intranasally infected under anesthesia (isoflurane/oxygen), with 2x10^5 cfu Bordetella pertussis B1917 in 40 µl Verweij medium at day 56.
Growth protocol Female BALB/c mice (Harlan, The Netherlands), 8-week-old, were divided in groups of three animals and housed in cages (macrolon III including filter top).
Extracted molecule total RNA
Extraction protocol RNA isolation was performed using a miRNeasy Mini Kit with DNAse treatment (Qiagen). RNA concentrations were determined using UV spectroscopy (Tech3 module, Synergy Mx, BioTek). RNA quality was determined using electrophoresis (RNA nano 6000 kit, 2100 Bioanalyzer, Agilent).
Label Cy3
Label protocol Per sample, 500 ng total RNA was amplified according to the Agilent QuickAmp kit manual (Agilent technologies). Amino-allyl modified nucleotides were incorporated during the aRNA synthesis (2.5 mM rGAU (GE Healthcare), 0.75 mM rCTP (GE Healthcare), 0.75 mM AA-rCTP (TriLink Biotechnologies). Synthesized aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit (Omega Bio-Tek). Test samples were labeled with Cy3 and a Reference sample (made by pooling equimolar amounts of RNA from Test samples) was labeled with Cy5. 5 µg of aRNA was dried down and dissolved in 50 mM carbonate buffer pH 8.5. Individual vials of Cy3/Cy5 from the mono-reactive dye packs (GE Healthcare) were dissolved in 200 µl DMSO. To each sample, 10 µl of the appropriate CyDye dissolved in DMSO was added and the mixture was incubated for 1h. Reactions were quenched with the addition of 5 µl 4M hydroxylamine (Sigma-Aldrich). The labeled aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit. The yields of aRNA and CyDye incorporation were measured on the NanoDrop ND-1000.
 
Channel 2
Source name lung
Organism Mus musculus
Characteristics sample type: common reference
Treatment protocol Three healthy mice were euthanized at day 0 and considered as naïve group. Mice assigned to the challenge groups were were intranasally infected under anesthesia (isoflurane/oxygen), with 2x10^5 cfu Bordetella pertussis B1917 in 40 µl Verweij medium at day 0. For both primary and secondary infections, mice were were intranasally infected under anesthesia (isoflurane/oxygen), with 2x10^5 cfu Bordetella pertussis B1917 in 40 µl Verweij medium at day 56.
Growth protocol Female BALB/c mice (Harlan, The Netherlands), 8-week-old, were divided in groups of three animals and housed in cages (macrolon III including filter top).
Extracted molecule total RNA
Extraction protocol RNA isolation was performed using a miRNeasy Mini Kit with DNAse treatment (Qiagen). RNA concentrations were determined using UV spectroscopy (Tech3 module, Synergy Mx, BioTek). RNA quality was determined using electrophoresis (RNA nano 6000 kit, 2100 Bioanalyzer, Agilent).
Label Cy5
Label protocol Per sample, 500 ng total RNA was amplified according to the Agilent QuickAmp kit manual (Agilent technologies). Amino-allyl modified nucleotides were incorporated during the aRNA synthesis (2.5 mM rGAU (GE Healthcare), 0.75 mM rCTP (GE Healthcare), 0.75 mM AA-rCTP (TriLink Biotechnologies). Synthesized aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit (Omega Bio-Tek). Test samples were labeled with Cy3 and a Reference sample (made by pooling equimolar amounts of RNA from Test samples) was labeled with Cy5. 5 µg of aRNA was dried down and dissolved in 50 mM carbonate buffer pH 8.5. Individual vials of Cy3/Cy5 from the mono-reactive dye packs (GE Healthcare) were dissolved in 200 µl DMSO. To each sample, 10 µl of the appropriate CyDye dissolved in DMSO was added and the mixture was incubated for 1h. Reactions were quenched with the addition of 5 µl 4M hydroxylamine (Sigma-Aldrich). The labeled aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit. The yields of aRNA and CyDye incorporation were measured on the NanoDrop ND-1000.
 
 
Hybridization protocol Each hybridization mixture was made up from 1.1 µg Test (Cy3) and 1.1 µg Reference (Cy5) sample. Samples were dried and 1.98 µl of the appropriate sample tracking control (STC, Roche Nimblegen) was added. The hybridization cocktail was made according to the manufacturer’s instructions (Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0, Roche Nimblegen). 5.22 µl from this mix was added to each sample. The samples were incubated for 5 min at 95°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto a 12x135k Mus musculus microarray (Catalog no. 05543797001, Design 090901 MM9 EXP HX12) containing 44,170 genes with 3 probes per target gene. Microarrays were hybridized for 20 hours at 42°C with the NimbleGen Hybridization System 4 (Roche Nimblegen). Afterwards, the slides were washed according to the Nimblegen Arrays User’s Guide – Gene Expression Arrays Version 5.0
Scan protocol Slides were scanned in an ozone-free room with a Agilent DNA microarray scanner G2565CA (Agilent Technologies). Feature extraction was performed with NimbleScan v2.5 (Roche Nimblegen).
Data processing Raw microarray signal data were normalised in R (www.r-project.org), using a four step approach: (1) natural log-transformation, (2) quantile normalisation of all scans, (3) correcting the sample spot signal for the corresponding reference spot signal and (4) averaging data from replicate oligo spots. Normalised data for the resulting 44170 oligonucleotides were further analysed in R and Microsoft Excel. Further calculations were based on logratios compared to uninfected animals.
 
Submission date Nov 27, 2015
Last update date Oct 09, 2016
Contact name Jeroen Pennings
E-mail(s) Jeroen.Pennings@rivm.nl
Phone +31 88 689 2214
Organization name Natl. Inst. Public Health & Environment
Street address A. van Leeuwenhoeklaan 9
City Bilthoven
ZIP/Postal code 3721MA
Country Netherlands
 
Platform ID GPL15887
Series (1)
GSE75438 Molecular signatures of infection-induced immunity against Bordetella pertussis.

Data table header descriptions
ID_REF
VALUE normalised data contains log2-ratios to the average of uninfected animals

Data table
ID_REF VALUE
AB000096 -0.051800583
AB000490 -0.292082844
AB001425 0.085299598
AB001435 -0.425302337
AB001539 -0.36795972
AB001750 1.655832615
AB001926 1.394916805
AB003502 0.711622754
AB004048 -0.856824201
AB005662 -0.289889489
AB005665 -0.114826083
AB005909 -0.062835803
AB006034 -0.436273444
AB006103 -0.515032153
AB007407 -0.550398369
AB008928 -0.33242588
AB009369 0.776560602
AB010088 -0.405415611
AB010122 -0.051212716
AB011499 0.086523389

Total number of rows: 44170

Table truncated, full table size 944 Kbytes.




Supplementary file Size Download File type/resource
GSM1955210_546506A09.ftr.gz 4.0 Mb (ftp)(http) FTR
Processed data included within Sample table

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