|
| Status |
Public on May 11, 2011 |
| Title |
fetal liver - trisomic - female - 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
fetal liver
|
| Organism |
Mus musculus |
| Characteristics |
tissue: fetal liver
genotype: Ts1Cje
|
| 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 fetal liver 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 |
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