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Sample GSM1289039 Query DataSets for GSM1289039
Status Public on Sep 02, 2014
Title lung 2 days [L2_30]
Sample type RNA
 
Channel 1
Source name lung 2 days
Organism Mus musculus
Characteristics strain: BALB/c
gender: female
age: 8 weeks
infected with: 2x10^5 cfu Bordetella pertussis B1917
tissue: lung
time after infection: 2 days
Treatment protocol Three healthy mice were euthanized at day 0 and considered as naïve group. Other mice were intranasally infected under anesthesia (isoflurane/oxygen), with 2x10^5 cfu Bordetella pertussis B1917 in 40 µl Verweij medium at day 0. Groups of animals (n=3) were euthanized after 2, 4, 6 hours, and after 1, 2, 4, 7, 10, 14, 21 and 28 days of infection, respectively. Please note that some timepoints were not used for microarray analysis, but were included to compare bacterial counts.
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 2 days (reference pool)
Organism Mus musculus
Characteristics strain: BALB/c
gender: female
age: 8 weeks
tissue: lung
Treatment protocol Three healthy mice were euthanized at day 0 and considered as naïve group. Other mice were intranasally infected under anesthesia (isoflurane/oxygen), with 2x10^5 cfu Bordetella pertussis B1917 in 40 µl Verweij medium at day 0. Groups of animals (n=3) were euthanized after 2, 4, 6 hours, and after 1, 2, 4, 7, 10, 14, 21 and 28 days of infection, respectively. Please note that some timepoints were not used for microarray analysis, but were included to compare bacterial counts.
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).
Description The signal#1 and signal#2 (i.e. SIGNAL_MEAN*.1 and SIGNAL_MEAN*.2) columns in .ftr files correspond to 'test/Cy3' and 'reference/Cy5' channels, respectively.
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. As the actual calculations for this experiment are done on ratios infected vs uninfected (t=0), the sample data table contains log2-ratios per sample vs average control. Further calculations were based on logratios compared to uninfected animals.
 
Submission date Dec 13, 2013
Last update date Sep 04, 2014
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)
GSE53294 Molecular signatures of the evolving immune response in mice following a Bordetella pertussis infection.

Data table header descriptions
ID_REF
VALUE log2-ratios per sample vs average control

Data table
ID_REF VALUE
AB000096 0.636786365
AB000490 -0.094705897
AB001425 0.179651245
AB001435 0.15517794
AB001539 0.051164825
AB001750 -0.450744676
AB001926 -0.225718735
AB003502 -0.671177619
AB004048 0.413209411
AB005662 0.159010614
AB005665 0.004091748
AB005909 0.030932724
AB006034 0.027803071
AB006103 -0.00347063
AB007407 0.061738529
AB008928 0.128516335
AB009369 -0.144954892
AB010088 0.01341831
AB010122 0.166841136
AB011499 -0.283996757

Total number of rows: 44170

Table truncated, full table size 938 Kbytes.




Supplementary file Size Download File type/resource
GSM1289039_530674A11.ftr.gz 4.0 Mb (ftp)(http) FTR
Processed data included within Sample table

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