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Sample GSM596938 Query DataSets for GSM596938
Status Public on May 11, 2011
Title placenta - control - male - 6
Sample type RNA
 
Channel 1
Source name placenta
Organism Mus musculus
Characteristics tissue: placenta
genotype: wild type
Treatment protocol Females were sacrificed on time point day E15.5 using CO2/O2. From pregnant mice the complete embryo strand was collected and every single embryo was processed further. Placenta and fetal liver were collected for RNA extraction and paws were collected for DNA extraction and genotyping. All tissues were immediately frozen in liquid nitrogen and stored at -80 °C until further processing.
Growth protocol To obtain Ts1Cje and wildtype fetuses for RNA isolation, female C3H/HeNHsd mice of 8-10 weeks of age were bred with male B6EiC3SnF1/J mice (control group) or male B6EiC3Sn-Ts(16C-tel)1Cje1 mice with genotype Ts/+ (Down group). After mating during the night, females were separated in the morning, at timepoint day E0.5, from the male and feasible vaginal plugs were scored.
Extracted molecule total RNA
Extraction protocol RNA was extracted from placenta and fetal liver using the miRNeasy kit (Qiagen). RNA concentrations were measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity of the RNA samples was investigated with the BioAnalyzer (Agilent Technolgies, Amstelveen, The Netherlands) using the RNA nano 6000 kit (Agilent Technologies) yielding RIN-values ≥ 9.6.
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 common placenta reference
Organism Mus musculus
Characteristics sample type: reference
Treatment protocol Females were sacrificed on time point day E15.5 using CO2/O2. From pregnant mice the complete embryo strand was collected and every single embryo was processed further. Placenta and fetal liver were collected for RNA extraction and paws were collected for DNA extraction and genotyping. All tissues were immediately frozen in liquid nitrogen and stored at -80 °C until further processing.
Growth protocol To obtain Ts1Cje and wildtype fetuses for RNA isolation, female C3H/HeNHsd mice of 8-10 weeks of age were bred with male B6EiC3SnF1/J mice (control group) or male B6EiC3Sn-Ts(16C-tel)1Cje1 mice with genotype Ts/+ (Down group). After mating during the night, females were separated in the morning, at timepoint day E0.5, from the male and feasible vaginal plugs were scored.
Extracted molecule total RNA
Extraction protocol RNA was extracted from placenta and fetal liver using the miRNeasy kit (Qiagen). RNA concentrations were measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity of the RNA samples was investigated with the BioAnalyzer (Agilent Technolgies, Amstelveen, The Netherlands) using the RNA nano 6000 kit (Agilent Technologies) yielding RIN-values ≥ 9.6.
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 {Baken, 2006 74 /id}: (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.
 
Submission date Sep 22, 2010
Last update date May 11, 2011
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 GPL10192
Series (1)
GSE24272 Biomarkers for Down Syndrome screening in murine fetal liver and placenta

Data table header descriptions
ID_REF
VALUE The tabel with normalised data contains untransformed signal intensities obtained by the R normalisation step.

Data table
ID_REF VALUE
AB000096 23044.62808
AB000490 526.3455302
AB001425 506.8927307
AB001435 481.6051839
AB001539 436.6138955
AB001750 6879.329709
AB001926 1610.570721
AB003502 9942.358074
AB004048 1688.877605
AB005662 474.112813
AB005665 324.4711551
AB005909 319.1062399
AB006034 337.3099664
AB006103 341.9202433
AB007407 404.0914281
AB008928 702.2217872
AB009369 422.7348257
AB010088 392.2447889
AB010122 1293.232757
AB011499 945.0343945

Total number of rows: 44170

Table truncated, full table size 918 Kbytes.




Supplementary file Size Download File type/resource
GSM596938_434026A01.ftr.gz 4.0 Mb (ftp)(http) FTR
Processed data included within Sample table

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